Coleopteran-resistant transgenic plants and methods of their production

ABSTRACT

Disclosed are nucleic acid segments comprising synthetically-modified genes encoding Coleopteran-toxic  B. thuringiensis  δ-endotoxins. Also disclosed are methods of using these genes for the recombinant expression of polypeptides, the preparation of vectors containing the genes, and methods for transforming suitable host cells.

1.0 BACKGROUND OF THE INVENTION

[0001] 1.1 Field of the Invention

[0002] This invention relates to transformed host cells and vectorswhich comprise nucleic acid segments encoding genetically-engineered,recombinant Bacillus thuringiensis δ-endotoxins which are active againstColeopteran insects.

[0003] 1.2 Description of the Related Art

[0004] Almost all field crops, plants, and commercial farming areas aresusceptible to attack by one or more insect pests. Particularlyproblematic are Coleopteran and Lepidoptern pests. For example,vegetable and cole crops such as artichokes, kohlrabi, arugula, leeks,asparagus, lentils, beans, lettuce (e.g., head, leaf, romaine), beets,bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon,crenshaw, honeydew,cantaloupe), brussels sprouts, cabbage, cardoni,carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas,parsnips, chicory, peas, chinese cabbage, peppers, collards, potatoes,cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga,eggplant, salsify, escarole, shallots, endive, soybean, garlic, spinach,green onions, squash, greens, sugar beets, sweet potatoes, turnip, swisschard, horseradish, tomatoes, kale, turnips, and a variety of spices aresensitive to infestation by one or more of the following insect pests:alfalfa looper, armyworm, beet armyworm, artichoke plume moth, cabbagebudworm, cabbage looper, cabbage webworm, corn earworm, celeryleafeater, cross-striped cabbageworm, european corn borer, diamondbackmoth, green cloverworm, imported cabbageworm, melonworm, omnivorousleafroller, pickleworm, rindworm complex, saltmarsh caterpillar, soybeanlooper, tobacco budworm, tomato fruitworm, tomato hornworm, tomatopinworm, velvetbean caterpillar, and yellowstriped armyworm. Likewise,pasture and hay crops such as alfalfa, pasture grasses and silage areoften attacked by such pests as armyworm, beef armyworm, alfalfacaterpillar, European skipper, a variety of loopers and webworms, aswell as yellowstriped armyworms.

[0005] Fruit and vine crops such as apples, apricots, cherries,nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts,filberts, pecans, pistachios, walnuts, citrus, blackberries,blueberries, boysenberries, cranberries, currants, loganberries,raspberries, strawberries, grapes, avocados, bananas, kiwi, persimmons,pomegranate, pineapple, tropical fruits are often susceptible to attackand defoliation by achema sphinx moth, amorbia, armyworm, citruscutworm, banana skipper, blackheaded fireworm, blueberry leafroller,cankerworm, cherry fruitworm, citrus cutworm, cranberry girdler, easterntent caterpillar, fall webworm, fall webworm, filbert leafroller,filbert webworm, fruit tree leafroller, grape berry moth, grapeleaffolder, grape leaf skeletonizer, green fruitworm,gummosos-batrachedra commosae, gypsy moth, hickory shuckworm, hornworms,loopers, navel orangeworm, obliquebanded leafroller, omnivorousleafroller. omnivorous looper, orange tortrix, orangedog, oriental fruitmoth, pandemis leafroller, peach twig borer, pecan nut casebearer,redbanded leafroller, redhumped caterpillar, roughskinned cutworm,saltmarsh caterpillar, spanworm, tent caterpillar, thecla-theclabasillides, tobacco budworm, tortrix moth, tufted apple budmoth,variegated leafroller, walnut caterpillar, western tent caterpillar, andyellowstriped armyworm.

[0006] Field crops such as canola/rape seed, evening primrose, meadowfoam, corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice,safflower, small grains (barley, oats, rye, wheat, etc.), sorghum,soybeans, sunflowers, and tobacco are often targets for infestation byinsects including armyworm, asian and other corn borers, bandedsunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm(including southern and western varieties), cotton leaf perforator,diamondback moth, european corn borer, green cloverworm, headmoth,headworm, imported cabbageworm, loopers (including Anacamptodes spp.),obliquebanded leafroller, omnivorous leaftier, podworm, podworm,saltmarsh caterpillar, southwestern corn borer, soybean looper, spottedcutworm, sunflower moth, tobacco budworm, tobacco hornworm, velvetbeancaterpillar,

[0007] Bedding plants, flowers, ornamentals, vegetables and containerstock are frequently fed upon by a host of insect pests such asarmyworm, azalea moth, beet armyworm, diamondback moth, ello moth(hornworm), Florida fern caterpillar, Io moth, loopers, oleander moth,omnivorous leafroller, omnivorous looper, and tobacco budworm.

[0008] Forests, fruit, ornamental, and nut-bearing trees, as well asshrubs and other nursery stock are often susceptible to attack fromdiverse insects such as bagworm, blackheaded budworm, browntail moth,california oakworm, douglas fir tussock moth, elm spanworm, fallwebworm, fruittree leafroller, greenstriped mapleworm, gypsy moth, jackpine budworm, mimosa webworm, pine butterfly, redhumped caterpillar,saddleback caterpillar, saddle prominent caterpillar, spring and fallcankerworm, spruce budworm, tent caterpillar, tortrix, and westerntussock moth. Likewise, turf grasses are often attacked by pests such asarmyworm, sod webworm, and tropical sod webworm.

[0009] Because crops of commercial interest are often the target ofinsect attack, environmentally-sensitive methods for controlling oreradicating insect infestation are desirable in many instances. This isparticularly true for farmers, nurserymen, growers, and commercial andresidential areas which seek to control insect populations usingeco-friendly compositions.

[0010] The most widely used environmentally-sensitive insecticidalformulations developed in recent years have been composed of microbialpesticides derived from the bacterium Bacillus thuringiensis. B.thuringiensis is a Gram-positive bacterium that produces crystalproteins or inclusion bodies which are specifically toxic to certainorders and species of insects. Many different strains of B.thuringiensis have been shown to produce insecticidal crystal proteins.Compositions including B. thuringiensis strains which produceinsecticidal proteins have been commercially-available and used asenvironmentally-acceptable insecticides because they are quite toxic tothe specific target insect, but are harmless to plants and othernon-targeted organisms.

[0011] 1.2.1 δ-Endotoxins

[0012] δ-endotoxins are used to control a wide range of leaf-eatingcaterpillars and beetles, as well as mosquitoes. These proteinaceousparasporal crystals, also referred to as insecticidal crystal proteins,crystal proteins, Bt inclusions, crystaline inclusions, inclusionbodies, and Bt toxins, are a large collection of insecticidal proteinsproduced by B. thuringiensis that are toxic upon ingestion by asusceptible insect host. Over the past decade research on the structureand function of B. thuringiensis toxins has covered all of the majortoxin categories, and while these toxins differ in specific structureand function, general similarities in the structure and function areassumed. Based on the accumulated knowledge of B. thuringiensis toxins,a generalized mode of action for B. thuringiensis toxins has beencreated and includes: ingestion by the insect, solubilization in theinsect midgut (a combination stomach and small intestine), resistance todigestive enzymes sometimes with partial digestion actually “activating”the toxin binding to the midgut cells, formation of a pore in the insectcells and the disruption of cellular homeostasis (English and Slatin,1992).

[0013] 1.2.2 Genes Encoding Crystal Proteins

[0014] Many of the δ-endotoxins are related to various degrees bysimilarities in their amino acid sequences. Historically, the proteinsand the genes which encode them were classified based largely upon theirspectrum of insecticidal activity. The review by Höfte and Whiteley(1989) discusses the genes and proteins that were identified in B.thuringiensis prior to 1990, and sets forth the nomenclature andclassification scheme which has traditionally been applied to B.thuringiensis genes and proteins: cryI genes encode lepidopteran-toxicCryI proteins. cryII genes encode CryII proteins that are toxic to bothlepidopterans and dipterans. cryIII genes encode coleopteran-toxicCryIII proteins, while cryIV genes encode dipteran-toxic CryIV proteins,etc. Based on the degree of sequence similarity, the proteins werefurther classified into subfamilies; more highly related proteins withineach family were assigned divisional letters such as CryIA, CryIB,CryIC, etc. Even more closely related proteins within each division weregiven names such as CryIC1, CryIC2, etc.

[0015] Recently a new nomenclature was developed which systematicallyclassifies the Cry proteins based upon amino acid sequence homologyrather than upon insect target specificities. This classificationscheme, including most of the known toxins but not including allelicvariations in individual polypeptides, is summarized in Table 1. TABLE 1KNOWN B. THURINGIENSIS δ-ENDOTOXINS, GENBANK ACCESSION NUMBERS, ANDREVISED NOMENCLATURE^(A) New Old GenBank Accession # Cry1Aa1 CryIA(a)M11250 Cry1Aa2 CryIA(a) M10917 Cry1Aa3 CryIA(a) D00348 Cry1Aa4 CryIA(a)X13535 Cry1Aa5 CryIA(a) D175182 Cry1Aa6 CryIA(a) U43605 Cry1Ab1 CryIA(b)M13898 Cry1Ab2 CryIA(b) M12661 Cry1Ab3 CryIA(b) M15271 Cry1Ab4 CryIA(b)D00117 Cry1Ab5 CryIA(b) X04698 Cry1Ab6 CryIA(b) M37263 Cry1Ab7 CryIA(b)X13233 Cry1Ab8 CryIA(b) M16463 Cry1Ab9 CryIA(b) X54939 Cry1Ab10 CryIA(b)A29125 Cry1Ac1 CryIA(c) M11068 Cry1Ac2 CryIA(c) M35524 Cry1Ac3 CryIA(c)X54159 Cry1Ac4 CryIA(c) M73249 Cry1Ac5 CryIA(c) M73248 Cry1Ac6 CryIA(c)U43606 Cry1Ac7 CryIA(c) U87793 Cry1Ac8 CryIA(c) U87397 Cry1Ac9 CryIA(c)U89872 Cry1Ac10 CryIA(c) AJ002514 Cry1Ad1 CryIA(d) M73250 Cry1Ae1CryIA(e) M65252 Cry1Ba1 CryIB X06711 Cry1Ba2 X95704 Cry1Bb1 ET5 L32020Cry1Bc1 CryIb(c) Z46442 Cry1Bd1 CryE1 U70726 Cry1Ca1 CryIC X07518Cry1Ca2 CryIC X13620 Cry1Ca3 CryIC M73251 Cry1Ca4 CryIC A27642 Cry1Ca5CryIC X96682 Cry1Ca6 CryIC X96683 Cry1Ca7 CryIC X96684 Cry1Cb1 CryIC(b)M97880 Cry1Da1 CryID X54160 Cry1Db1 PrtB Z22511 Cry1Ea1 CryIE X53985Cry1Ea2 CryIE X56144 Cry1Ea3 CryIE M73252 Cry1Ea4 U94323 Cry1Eb1CryIE(b) M73253 Cry1Fa1 CryIF M63897 Cry1Fa2 CryIF M63897 Cry1Fb1 PrtDZ22512 Cry1Ga1 PrtA Z22510 Cry1Ga2 CryIM Y09326 Cry1Gb1 CryH2 U70725Cry1Ha1 PrtC Z22513 Cry1Hb1 U35780 Cry1Ia1 CryV X62821 Cry1Ia2 CryVM98544 Cry1Ia3 CryV L36338 Cry1Ia4 CryV L49391 Cry1Ia5 CryV Y08920Cry1Ib1 CryV U07642 Cry1Ja1 ET4 L32019 Cry1Jb1 ET1 U31527 Cry1Ka1 U28801Cry2Aa1 CryIIA M31738 Cry2Aa2 CryIIA M23723 Cry2Aa3 D86084 Cry2Ab1CryIIB M23724 Cry2Ab2 CryIIB X55416 Cry2Ac1 CryIIC X57252 Cry3Aa1CryIIIA M22472 Cry3Aa2 CryIIIA J02978 Cry3Aa3 CryIIIA Y00420 Cry3Aa4CryIIIA M30503 Cry3Aa5 CryIIIA M37207 Cry3Aa6 CryIIIA U10985 Cry3Ba1CryIIIB X17123 Cry3Ba2 CryIIIB A07234 Cry3Bb1 CryIIIB2 M89794 Cry3Bb2CryIIIC(b) U31633 Cry3Ca1 CryIIID X59797 Cry4Aa1 CryIVA Y00423 Cry4Aa2CryIVA D00248 Cry4Ba1 CryIVB X07423 Cry4Ba2 CryIVB X07082 Cry4Ba3 CryIVBM20242 Cry4Ba4 CryIVB D00247 Cry5Aa1 CryVA(a) L07025 Cry5Ab1 CryVA(b)L07026 Cry5Ba1 PS86Q3 U19725 Cry6Aa1 CryVIA L07022 Cry6Ba1 CryVIB L07024Cry7Aa1 CryIIIC M64478 Cry7Ab1 CryIIICb U04367 Cry8Aa1 CryIIIE U04364Cry8Ba1 CryIIIG U04365 Cry8Ca1 CryIIIF U04366 Cry9Aa1 CryIG X58120Cry9Aa2 CryIG X58534 Cry9Ba1 CryIX X75019 Cry9Ca1 CryIH Z37527 Cry9Da1N141 D85560 Cry10Aa1 CryIVC M12662 Cry11Aa1 CryIVD M31737 Cry11Aa2CryIVD M22860 Cry11Ba1 Jeg80 X86902 Cry12Aa1 CryVB L07027 Cry13Aa1 CryVCL07023 Cry14Aa1 CryVD U13955 Cry15Aa1 34 kDa M76442 Cry16Aa1 cbm71X94146 Cry17Aa1 cbm71 X99478 Cry18Aa1 CryBP1 X99049 Cry19Aa1 Jeg65Y08920 Cry20Aa1 U82518 Cry21Aa1 I32932 Cry22Aa1 I34547 Cyt1Aa1 CytAX03182 Cyt1Aa2 CytA X04338 Cyt1Aa3 CytA Y00135 Cyt1Aa4 CytA M35968Cyt1Ab1 CytM X98793 Cyt1Ba1 U37196 Cyt2Aa1 CytB Z14147 Cyt2Ba1 “CytB”U52043 Cyt2Ba2 “CytB” AF020789 Cyt2Ba3 “CytB” AF022884 Cyt2Ba4 “CytB”AF022885 Cyt2Ba5 “CytB” AF022886 Cyt2Bb1 U82519

[0016] 1.2.3 Bioinsecticide Polypeptide Compositions

[0017] The utility of bacterial crystal proteins as insecticides wasextended beyond lepidopterans and dipteran larvae when the firstisolation of a coleopteran-toxic B. thuringiensis strain was reported(Krieg et al., 1983; 1984). This strain (described in U.S. Pat. No.4,766,203, specifically incorporated herein by reference), designated B.thuringiensis var. tenebrionis, is reported to be toxic to larvae of thecoleopteran insects Agelastica alni (blue alder leaf beetle) andLeptinotarsa decemlineata (Colorado potato beetle).

[0018] U.S. Pat. No. 5,024,837 also describes hybrid B. thuringiensisvar. kurstaki strains which showed activity against lepidopteraninsects. U.S. Pat. No. 4,797,279 (corresponding to EP 0221024) disclosesa hybrid B. thuringiensis containing a plasmid from B. thuringiensisvar. kurstaki encoding a lepidopteran-toxic crystal protein-encodinggene and a plasmid from B. thuringiensis tenebrionis encoding acoleopteran-toxic crystal protein-encoding gene. The hybrid B.thuringiensis strain produces crystal proteins characteristic of thosemade by both B. thuringiensis kurstaki and B. thuringiensis tenebrionis.U.S. Pat. No. 4,910,016 (corresponding to EP 0303379) discloses a B.thuringiensis isolate identified as B. thuringiensis MT 104 which hasinsecticidal activity against coleopterans and lepidopterans.

[0019] 1.2.4 Molecular Genetic Techniques Facilitate Protein Engineering

[0020] The revolution in molecular genetics over the past decade hasfacilitated a logical and orderly approach to engineering proteins withimproved properties. Site specific and random mutagenesis methods, theadvent of polymerase chain reaction (PCR™) methodologies, and relatedadvances in the field have permitted an extensive collection of toolsfor changing both amino acid sequence, and underlying genetic sequencesfor a variety of proteins of commercial, medical, and agriculturalinterest.

[0021] Following the rapid increase in the number and types of crystalproteins which have been identified in the past decade, researchersbegan to theorize about using such techniques to improve theinsecticidal activity of various crystal proteins. In theory,improvements to δ-endotoxins should be possible using the methodsavailable to protein engineers working in the art, and it was logical toassume that it would be possible to isolate improved variants of thewild-type crystal proteins isolated to date. By strengthening one ormore of the aforementioned steps in the mode of action of the toxin,improved molecules should provide enhanced activity, and therefore,represent a breakthrough in the field. If specific amino acid residueson the protein are identified to be responsible for a specific step inthe mode of action, then these residues can be targeted for mutagenesisto improve performance

[0022] 1.2.5 Structural Analyses of Crystal Proteins

[0023] The combination of structural analyses of B. thuringiensis toxinsfollowed by an investigation of the function of such structures, motifs,and the like has taught that specific regions of crystal proteinendotoxins are, in a general way, responsible for particular functions.

[0024] Domain 1, for example, from Cry3Bb and Cry1Ac has been found tobe responsible for ion channel activity, the initial step in formationof a pore (Walters et al., 1993; Von Tersch et al., 1994). Domains 2 and3 have been found to be responsible for receptor binding andinsecticidal specificity (Aronson et al., 1995; Caramori et al., 1991;Chen et al. 1993; de Maagd et al., 1996; Ge et al., 1991; Lee et al.,1992; Lee et al., 1995; Lu et al., 1994; Smedley and Ellar, 1996; Smithand Ellar, 1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Wu andDean, 1996). Regions in domain 2 and 3 can also impact the ion channelactivity of some toxins (Chen et al., 1993, Wolfersberger et al., 1996;Von Tersch et al., 1994).

[0025] 1.3 Deficiencies in the Prior Art

[0026] Unfortunately, while many laboratories have attempted to makemutated crystal proteins, few have succeeded in making mutated crystalproteins with improved lepidopteran toxicity. In almost all of theexamples of genetically-engineered B. thuringiensis toxins in theliterature, the biological activity of the mutated crystal protein is nobetter than that of the wild-type protein, and in many cases, theactivity is decreased or destroyed altogether (Almond and Dean, 1993;Aronson et al., 1995; Chen et al., 1993, Chen et al., 1995; Ge et al.,1991; Kwak et al., 1995; Lu et al., 1994; Rajamohan et al., 1995;Rajamohan et al., 1996; Smedley and Ellar, 1996; Smith and Ellar, 1994;Wolfersberger et al., 1996; Wu and Aronson, 1992).

[0027] For a crystal protein having approximately 650 amino acids in thesequence of its active toxin, and the possibility of 20 different aminoacids at each position in this sequence, the likelihood of arbitrarilycreating a successful new structure is remote, even if a generalfunction to a stretch of 250-300 amino acids can be assigned. Indeed,the above prior art with respect to crystal protein gene mutagenesis hasbeen concerned primarily with studying the structure and function of thecrystal proteins, using mutagenesis to perturb some step in the mode ofaction, rather than with engineering improved toxins.

[0028] Collectively, the limited successes in the art to developsynthetic toxins with improved insecticidal activity have stifledprogress in this area and confounded the search for improved endotoxinsor crystal proteins. Rather than following simple and predictable rules,the successful engineering of an improved crystal protein may involvedifferent strategies, depending on the crystal protein being improvedand the insect pests being targeted. Thus, the process is highlyempirical.

[0029] Accordingly, traditional recombinant DNA technology is clearlynot routine experimentation for providing improved insecticidal crystalproteins. What are lacking in the prior art are rational methods forproducing genetically-engineered B. thuringiensis crystal proteins thathave improved insecticidal activity and, in particular, improvedtoxicity towards a wide range of lepidopteran insect pests.

2.0 SUMMARY OF THE INVENTION

[0030] The present invention seeks to overcome these and other drawbacksinherent in the prior art by providing genetically-engineered modifiedB. thuringiensis δ-endotoxins (Cry*), and in particular modified Cry3δ-endotoxins (designated Cry3* endotoxins). Also provided are nucleicacid sequences comprising one or more genes which encode such modifiedproteins. Particularly preferred genes include cry3* genes such as cry3A*, cry3B *, and cry3C* genes, particularly cry3B* genes, and moreparticularly, cry3Bb* genes, that encode modified crystal proteinshaving improved insecticidal activity against target pests.

[0031] Also disclosed are novel methods for constructing synthetic Cry3*proteins, synthetically-modified nucleic acid sequences encoding suchproteins, and compositions arising therefrom. Also provided aresynthetic cry3* expression vectors and various methods of using theimproved genes and vectors. In a preferred embodiment, the inventiondiscloses and claims Cry3B* proteins and cry3B* genes which encodeimproved insecticidal polypeptides.

[0032] In preferred embodiments, channel-forming toxin design methodsare disclosed which have been used to produce a specific set of designedCry3Bb* toxins with improved biological activity. These improved Cry3Bb*proteins are listed in Table 2 along with their respective amino acidchanges from wild-type (WT) Cry3Bb, the nucleotide changes present inthe altered cry3Bb* gene encoding the protein, the fold increase inbioactivity over WT Cry3Bb, the structural site of the alteration, andthe design method(s) used to create the new toxins.

[0033] Accordingly, the present invention provides in an overall andgeneral sense, mutagenized Cry3 protein-encoding genes and methods ofmaking and using such genes. As used herein the term “mutagenized cry3gene(s)” means one or more cry3 genes that have been mutagenized oraltered to contain one or more nucleotide sequences which are notpresent in the wild type sequences, and which encode mutant Cry3 crystalproteins (Cry3*) showing improved insecticidal activity. Suchmutagenized cry3 genes have been referred to in the Specification ascry3* genes. Exemplary cry3* genes include cry3A*, cry3B*, and cry3C*genes.

[0034] Exemplary mutagenized Cry3 protein-encoding genes include cry3Bgenes. As used herein the term “mutagenized cry3B gene(s)” means one ormore genes that have been mutagenized or altered to contain one or morenucleotide sequences which are not present in the wild type sequences,and which encode mutant Cry3B crystal proteins (Cry3B*) showing improvedinsecticidal activity. Such genes have been designated cry3B* genes.Exemplary cry3B* genes include cry3Ba* and cry3Bb* genes, which encodeCry3Ba* and Cry3Bb* proteins, respectively.

[0035] Likewise, the present invention provides mutagenized Cry3Aprotein-encoding genes and methods of making and using such genes. Asused herein the term “mutagenized cry3A gene(s)” means one or more genesthat have been mutagenized or altered to contain one or more nucleotidesequences which are not present in the wild type sequences, and whichencode mutant Cry3A crystal proteins (Cry3A*) showing improvedinsecticidal activity. Such mutagenized genes have been designated ascry3A* genes.

[0036] In similar fashion, the present invention provides mutagenizedCry3C protein-encoding genes and methods of making and using such genes.As used herein the term “mutagenized cry3C gene(s)” means one or moregenes that have been mutagenized or altered to contain one or morenucleotide sequences which are not present in the wild type sequences,and which encode mutant Cry3C crystal proteins (Cry3C*) showing improvedinsecticidal activity. Such mutagenized genes have been designated ascry3C* genes.

[0037] Preferably the novel sequences comprise nucleic acid sequences inwhich at least one, and preferably, more than one, and most preferably,a significant number, of wild-type cry3 nucleotides have been replacedwith one or more nucleotides, or where one or more nucleotides have beenadded to or deleted from the native nucleotide sequence for the purposeof altering, adding, or deleting the corresponding amino acids encodedby the nucleic acid sequence so mutagenized. The desired result,therefore, is alteration of the amino acid sequence of the encodedcrystal protein to provide toxins having improved or altered activityand/or specificity compared to that of the unmodified crystal protein.

[0038] Examples of preferred Cry2Bb*-encoding genes include cry3Bb.60,cry3Bb.11221, cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225,cry3Bb.11226. cry3Bb.11227, cry3Bb.11228, cry3Bb.11229, cry3Bb.11230,cry3Bb.11231, cry3Bb.11232, cry3Bb.11233, cry3Bb.11234, cry3Bb.11235,cry3Bb.11236, cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241,cry3Bb.11242, cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046,cry3Bb.11048, cry3Bb.11051, cry3Bb.11057, cry3Bb.11058, cry3Bb.11081,cry3Bb. 11082, cry3Bb.11083, cry3Bb.11084, cry3Bb.11095, andcry3Bb.11098. TABLE 2 CRY3BB* PROTEINS EXHIBITING IMPROVED ACTIVITYAGAINST SCRW LARVAE Cry3Bb* cry3Bb* Structural Fold Design ProteinPlasmid cry3Bb* Nucleotide Sequence Cry3Bb* Amino Site Increase OverMethod Designation Designation Changes Acid Changes of Changes WTActivity Used Cry3Bb.60 — — Δ1-159 Δα1-α3 3.6× 1, 6, 8 Cry3Bb.11221pEG1707 A460T, C461T, A462T, C464A, T154F, P155H, 1α3, 4 6.4× 1, 8T465C, T466C, T467A, A468T, L156H, L158R A469T, G470C, T472C, T473G,G474T, A477T, A478T, G479C Cry3Bb.11222 pEG1708 T687C, T688C, A689T,C691A, Y230L, H231S α6 4.0× 3, 7 A692G Cry3Bb.11223 pEG1709 T667C,T687C, T688A, A689G, S223P, Y230S α6 2.8× 3 C691A, A692G Cry3Bb.11224pEG1710 T687C, A692G H231R α6 5.0× 7, 8 Cry3Bb.11225 pEG1711 T687C,C691A H231N, T241S α6 3.6× 7 Cry3Bb.11226 pEG1712 T687C, C691A, A692C,T693C H231T α6 3.0× 7, 8 Cry3Bb.11227 pEG1713 C868A, G869A, G870T R290N1α7, β1 1.9× 2, 3, 4, 6 Cry3Bb.11228 pEGI714 C932T, A938C, T942G, G949A,S311L, N313T, 1β1, α8 4.1× 2, 4 T954C E317K Cry3Bb.11229 pEG1715 T931A,A933C, T942A, T945A, S311T, E317K, 1β1, α8 2.5× 2, 4 G949A, A953G, T954CY318C Cry3Bb.11230 pEG1716 T931G, A933C, C934G, T945G, S311A, L312V,1β1, α8 4.7× 2, 4 8 C946T, A947G, G951A, T954C Q316W Cry3Bb.11231pEG1717 T687C, A692G, C932T, A938C, H231R, S311L, α6; 1β1, α8 7.9× 2, 4,7, 8, T942G, G949A, T954C N313T, E317K 10 Cry3Bb.11232 pEG1718 T931A,A933G, T935C, T936A, S311T, L312P, 1β1, α8 5.1× 4 A938C, T939C, T942C,T945A, N313T, E317N G951T, T954C Cry3Bb.11233 pEG1719 T931G, A933C,T936G, T942C, S311A, Q316D 1β1, α8 2.2× 2, 4 C943T, T945A, C946G, G948C,T954C Cry3Bb.11234 pEG1720 T861C, T866C, C868A, T871C, 1289T, L291R,1α7, β1 4.1× 4 T872G, A875T, T877A, C878G, Y292F, S293R A882GCry3Bb.11235 pEG1721 T687C, A692G, C932T H231R, S311L α6; 1β1, α8 3.2×2, 4, 7, 8, 10 Cry3Bb.11236 pEG1722 T931A, C932T, A933C, T936C, S311I1β1, α8 3.1× 2, 4 T942G, T945A, T954C Cry3Bb.11237 pEG1723 T931A, C932T,A933C, T936C, S311I, N313H 1β1, α8 5.4× 2, 4 A937G, A938T, C941A, T942C,T945A, C946A, A947T, A950T, T954C Cry3Bb.11238 pEG1724 A933C, T936C,A937G, A938T, N313V, T314N, 1β1, α8 2.6× 2, 4 C941A, T942C, T945A,C946A, Q316M, E317V A947T, A950T, T954C Cry3Bb.11239 pEG1725 A933T,A938G, T939G, T942A, N313R, L315P, 1β1, α8 2.8× 2, 4 T944C, T945A,A947T, G948T, Q316L, E317A A950C, T954C Cry3Bb.11241 pEG1726 A860T,T861C, G862A, C868T, Y287F, D288N, 1α7, β1 2.6× 2, 3, 4, 6 G869T, T871C,A873T, T877A, R290L C878G, A879T Cry3Bb.11242 pEG1727 C868G, G869T R290V1α7, β1 2.5× 2, 3, 4, 6, 8 Cry3Bb.11032 pEG1041 A494G D165G α4 3.1× 2,4, 8 Cry3Bb.11035 pEG1046 G479A, A481C, A482C, S160N, K161P, α4 2.7× 8A484C, G485A, A486C, A494G P162H, D165G Cry3Bb.11036 pEG1047 A865G,T877C I289V, S293P 1α7, β1 4.3× 4 Cry3Bb.11046 pEG1052 G479A, A481C,A482C, S160N, K161P, α4; 1α7, β1 2.6× 2, 4, 8, 10 A484C, G485A, A486C,P162H, D165G, A494G, A865G, T877C I289V, S293P Cry3Bb.11048 pEG1054T309A, Δ310, Δ311, Δ312 D103E, ΔA104 1α2a, 2b 4.3× 8 Cry3Bb.11051pEG1057 A565G, A566G K189G 1α4, 5 3.0× 2, 3, 4 Cry3Bb.11057 pEG1062T309A, Δ310, Δ311, Δ312, D103E, ΔA104, 1α2a, 2b; α4 3.4× 2, 4, 8, 10G479A, A481C, A482C, S160N, K161P, A484C, G485A, A486C, A494G P162H,D165G Cry3Bb.11058 pEG1063 T309A, Δ310, Δ311, Δ312, D103E, ΔA104, 1α2a,2b; 3.5× 1, 8, 10 A460T, C461T, A462T, C464A, T154F, P155H, 1α3, 4T465C, T466C, T467A, A468T, L156H, L158R A469T, G470C, T472C, T473G,G474T, A477T, A478T, G479C Cry3Bb.11081 pEG1084 A494G, T931A, A933C,D165G, S311T, α4; 1β1, α8 6.1× 2, 4, 8, 10 T942A, T945A, G949A, T954CE317K Cry3Bb.11082 pEG1085 A494G, A865G, T877C, T914C, D165G, I289V, α4;1α7, β1; 4.9× 2, 4, 5, 8, β1; T931G, A933C, C934G, T945G, S293P, F305S,1β1, α8; β2; 9, 10 C946T, A947G, G951A, T954C, S311A, L312V, β3b A1043G,T1094C Q316W, Q348R, V365A Cry3Bb.11083 pEG1086 A865G, T877C, A1043GI289V, S293P, 1α7, β1; β2 7.4× 4, 5, 9, 10 Q348R Cry3Bb.11084 pEG1087A494G, C932T D165G, S311L α4; 1β1, α8 7.2× 2, 4, 8, 10 Cry3Bb.11095pEG1095 A1043G Q348R β2 4.6× 5, 9 Cry3Bb.11098 pEG1098 A494G, T687C,A692G, C932T, D165G, H231R, α4; α6, 1β1, 7.9× 2, 4, 7, 8 A938C, T942G,G949A, T954C S311L, N313T, α8 E317K

[0039] In a variety of illustrative embodiments, the inventors haveshown remarkable success in generating toxins with improved insecticidalactivity using these methods. In particular, the inventors haveidentified unique methods of analyzing and designing toxins havingimproved or enhanced insecticidal properties both in vitro and in vivo.

[0040] In addition to modifications of Cry3Bb peptides, those havingbenefit of the present teaching are now also able to make mutations in avariety of channel-forming toxins, and particularly in crystal proteinswhich are related to Cry3Bb either functionally or structurally. Infact, the inventors contemplate that any B. thuringiensis crystalprotein or peptide can be analyzed using the methods disclosed hereinand may be altered using the methods disclosed herein to produce crystalproteins having improved insecticidal specificity or activity.Alternatively, the inventors contemplate that those of skill in the arthaving the benefit of the teachings disclosed herein will be able toprepare not only mutated Cry3 toxins with improved activity, but alsoother crystal proteins including all of those proteins identified inTable 1, herein. In particular, the inventors contemplate the creationof Cry3* variants using one or more of the methods disclosed herein toproduce toxins with improved activity. For example, the inventors noteCry3A, Cry3B, and Cry3C crystal proteins (which are known in the art)may be modified using one or more of the design strategies employedherein, to prepare synthetically-modifiedcrystal proteins with improvedproperties. Likewise, one of skill in the art will even be able toutilize the teachings of the present disclosure to modify other channelforming toxins, including channel forming toxins other than B.thuringiensis crystal proteins, and even to modify proteins and channeltoxins not yet described or characterized.

[0041] Because the structures for insecticidal crystal proteins show aremarkable conservation of protein tertiary structure (Grochulski etal., 1995), and because many crystal proteins show significant aminoacid sequence identity to the Cry3Bb amino acid sequence within domain1, including proteins of the Cry1, Cry2, Cry3, Cry4, Cry5, Cry7, Cry8,Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, and Cry16 classes (Table 1),now in light of the inventors' surprising discovery, for the first time,those of skill in the art having benefit of the teachings disclosedherein will be able to broadly apply the methods of the invention tomodifying a host of crystal proteins with improved activity or alteredspecificity. Such methods will not only be limited to the insecticidalcrystal proteins disclosed in Table 1, but may also been applied to anyother related crystal protein, including those yet to be identified.

[0042] In particular, the high degree of homology between Cry3A, Cry3B,and Cry3C proteins is evident in the alignment of the primary amino acidsequence of the three proteins (FIG. 17A, FIG. 17B, and FIG. 17C).

[0043] As such, the disclosed methods may be now applied to preparationof modified crystal proteins having one or more alterations introducedusing one or more of the mutational design methods as disclosed herein.The inventors further contemplate that regions may be identified in oneor more domains of a crystal protein, or other channel forming toxinwhich may be similarly modified through site-specific or randommutagenesis to generate toxins having improved activity, oralternatively, altered specificity.

[0044] In certain applications, the creation of altered toxins havingincreased activity against one or more insects is desired.Alternatively, it may be desirable to utilize the methods describedherein for creating and identifying altered insecticidal crystalproteins which are active against a wider spectrum of susceptibleinsects. The inventors further contemplate that the creation of chimericinsecticidal crystal proteins comprising one or more of these mutationsmay be desirable for preparing “super” toxins which have the combinedadvantages of increased insecticidal activity and concomitant broadspectrum activity.

[0045] In light of the present disclosure, the mutagenesis of one ormore codons within the sequence of a toxin may result in the generationof a host of related insecticidal proteins having improved activity.While exemplary mutations have been described for each of the designstrategies employed in the present invention, the inventors contemplatethat mutations may also be made in insecticidal crystal proteins,including the loop regions, helices regions, active sites of the toxins,regions involved in protein oligomerization, and the like, which willgive rise to functional bioinsecticidal crystal proteins. All suchmutations are considered to fall within the scope of this disclosure.

[0046] In one illustrative embodiment, mutagenized cry3Bb* genes areobtained which encode Cry3Bb* variants that are generally based upon thewild-type Cry3Bb sequence, but that have one or more changesincorporated into the amino acid sequence of the protein using one ormore of the design strategies described and claimed herein.

[0047] In these and other embodiments, the mutated genes encoding thecrystal proteins may be modified so as to change about one, two, three,four, or five or so amino acids in the primary sequence of the encodedpolypeptide. Alternatively even more changes from the native sequencemay be introduced, such that the encoded protein may have at least about1% or 2%, or alternatively about 3% or about 4%, or even about 5% toabout 10%, or about 10% to about 15%, or even about 15% to about 20% ormore of the codons either altered, deleted, or otherwise modified. Incertain situations, it may even be desirable to alter substantially moreof the primary amino acid sequence to obtain the desired modifiedprotein. In such cases the inventors contemplate that from about 25%, toabout 50%, or even from about 50% to about 75%, or more of the native(or wild-type) codons either altered, deleted, or otherwise modified.Alternatively, mutations in the amino acid sequences or underlying DNAgene sequences which result in the insertion or deletion of one or moreamino acids within one or more regions of the crystal protein orpeptide.

[0048] To effect such changes in the primary sequence of the encodedpolypeptides, it may be desirable to mutate or delete one or morenucleotides from the nucleic acid sequences of the genes encoding suchpolypeptides, or alternatively, under certain circumstances to add oneor more nucleotides into the primary nucleic acid sequence at one ormore sites in the sequence. Frequently, several nucleotide residues maybe altered to produce the desired polypeptide. As such, the inventorscontemplate that in certain embodiments it may be desirable to alteronly one, two, three, four, or five or so nucleotides in the primarysequence. In other embodiments, which more changes are desired, themutagenesis may involve changing, deleting, or inserting 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or even 20 or so nucleotide residuesin the gene sequence. In still other embodiments, one may desire tomutate, delete, or insert 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, or even 90-100, 150, 200, 250, 300,350, 400, 450, or more nucleotides in the sequence of the gene in orderto prepare a cry3* gene which produces a Cry3* polypeptide having thedesired characteristics. In fact, any number of mutations, deletions,and/or insertions may be made in the primary sequence of the gene, solong as the encoded protein has the improved insecticidal activity orspecificity characteristics described herein.

[0049] Changing a large number of the codons in the nucleotide sequenceof an endotoxin-encoding gene may be particularly desirable and oftennecessary to achieve the desired results, particularly in the situationof “plantizing” a DNA sequence in order to express a DNA of non-plantorigin in a transformed plant cell. Such methods are routine to those ofskill in the plant genetics arts, and frequently many residues of aprimary gene sequence will be altered to facilitate expression of thegene in the plant cell. Preferably, the changes in the gene sequenceintroduce no changes in the amino acid sequence, or introduce onlyconservative replacements in the amino acid sequence such that thepolypeptide produced in the plant cell from the “plantized” nucleotidesequence is still fully functional, and has the desired qualities whenexpressed in the plant cell.

[0050] Genes and encoded proteins mutated in the manner of the inventionmay also be operatively linked to other protein-encoding nucleic acidsequences, or expressed as fusion proteins. Both N-terminal andC-terminal fusion proteins are contemplated. Virtually any protein- orpeptide-encoding DNA sequence, or combinations thereof, may be fused toa mutated cry3* sequence in order to encode a fusion protein. Thisincludes DNA sequences that encode targeting peptides, proteins forrecombinant expression, proteins to which one or more targeting peptidesis attached, protein subunits, domains from one or more crystalproteins, and the like. Such modifications to primary nucleotidesequences to enhance, target, or optimize expression of the genesequence in a particular host cell, tissue, or cellular localization,are well-known to those of skill in the art of protein engineering andmolecular biology, and it will be readily apparent to such artisans,having benefit of the teachings of this specification, how to facilitatesuch changes in the nucleotide sequence to produce the polypeptides andpolynucleotides disclosed herein.

[0051] In one aspect, the invention discloses and claims host cellscomprising one or more of the modified crystal proteins disclosedherein, and in particular, cells of B. thuringiensis strains EG11221,EG11222, EG11223, EG11224, EG11225, EG11226, EG11227, EG11228, EG11229,EG11230, EG11231, EG11232, EG11233, EG11234, EG11235, EG11236, EG11237,EG11238, EG11239, EG11241, EG11242, EG11032, EG11035, EG11036, EG11046,EG11048, EG11051, EG11057, EG11058, EG11081, EG11082, EG11083, EG11084,EG11095, and EG11098 which comprise recombinant DNA segments encodingsynthetically-modified Cry3 Bb* crystal proteins which demonstratesimproved insecticidal activity.

[0052] Likewise, the invention also discloses and claims cell culturesof B. thuringiensis EG11221, EG11222, EG11223, EG11224, EG11225,EG11226, EG11227, EG11228, EG11229, EG11230, EG11231, EG11232, EG11233,EG11234, EG11235, EG11236, EG11237, EG11238, EG11239, EG11241, EG11242,EG11032, EG11035, EG11036, EG11046, EG11048, EG11051, EG11057, EG11058,EG11081, EG11082, EG11083, EG11084, and EG11095, and 11098.

[0053] Such cell cultures may be biologically-pure cultures consistingof a single strain, or alternatively may be cell co-cultures consistingof one or more strains. Such cell cultures may be cultivated underconditions in which one or more additional B. thuringiensis or otherbacterial strains are simultaneously co-cultured with one or more of thedisclosed cultures, or alternatively, one or more of the cell culturesof the present invention may be combined with one or more additional B.thuringiensis or other bacterial strains following the independentculture of each. Such procedures may be useful when suspensions of cellscontaining two or more different crystal proteins are desired.

[0054] The subject cultures have been deposited under conditions thatassure that access to the cultures will be available during the pendencyof this patent application to one determined by the Commissioner ofPatents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and35 U.S.C. §122. The deposits are available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

[0055] Further, the subject culture deposits will be stored and madeavailable to the public in accord with the provisions of the BudapestTreaty for the Deposit of Microorganisms, i.e., they will be stored withall the care necessary to keep them viable and uncontaminated for aperiod of at least five years after the most recent request for thefinishing of a sample of the deposit, and in any case, for a period ofat least 30 (thirty) years after the date of deposit or for theenforceable life of any patent which may issue disclosing the cultures.The depositor acknowledges the duty to replace the deposits should thedepository be unable to furnish a sample when requested, due to thecondition of the deposits. All restrictions on the availability to thepublic of the subject culture deposits will be irrevocably removed uponthe granting of a patent disclosing them.

[0056] Cultures shown in Table 3 were deposited in the permanentcollection of the Agricultural Research Service Culture Collection,Northern Regional Research Laboratory (NRRL) under the terms of theBudapest Treaty. TABLE 3 STRAINS OF THE PRESENT INVENTION DEPOSITEDUNDER THE TERMS OF THE BUDAPEST TREATY Accession Number Strain DepositDate Protein (NRRL Number) EG11032 May 27, 1997 Cry3Bb.11032 B-21744EG11035 May 27, 1997 Cry3Bb.11035 B-21745 EG11036 May 27, 1997Cry3Bb.11036 B-21746 EG11037 May 27, 1997 Cry3Bb.11037 B-21747 EG11046May 27, 1997 Cry3Bb.11046 B-21748 EG11048 May 27, 1997 Cry3Bb.11048B-21749 EG11051 May 27, 1997 Cry3Bb.11051 B-21750 EG11057 May 27, 1997Cry3Bb.11057 B-21751 EG11058 May 27, 1997 Cry3Bb.11058 B-21752 EG11081May 27, 1997 Cry3Bb.11081 B-21753 EG11082 May 27, 1997 Cry3Bb.11082B-21754 EG11083 May 27, 1997 Cry3Bb.11083 B-21755 EG11084 May 27, 1997Cry3Bb.11084 B-21756 EG11095 May 27, 1997 Cry3Bb.11095 B-21757 EG11204May 27, 1997 Cry3Bb.11204 B-21758 EG11221 May 27, 1997 Cry3Bb.11221B-21759 EG11222 May 27, 1997 Cry3Bb.11222 B-21760 EG11223 May 27, 1997Cry3Bb.11223 B-21761 EG11224 May 27, 1997 Cry3Bb.11224 B-21762 EG11225May 27, 1997 Cry3Bb.11225 B-21763 EG11226 May 27, 1997 Cry3Bb.11226B-21764 EG11227 May 27, 1997 Cry3Bb.11227 B-12765 EG11228 May 27, 1997Cry3Bb.11228 B-12766 EG11229 May 27, 1997 Cry3Bb.11229 B-21767 EG11230May 27, 1997 Cry3Bb.11230 B-21768 EG11231 May 27, 1997 Cry3Bb.11231B-21769 EG11232 May 27, 1997 Cry3Bb.11232 B-12770 EG11233 May 27, 1997Cry3Bb.11233 B-21771 EG11234 May 27, 1997 Cry3Bb.11234 B-21772 EG11235May 27, 1997 Cry3Bb.11235 B-21773 EG11236 May 27, 1997 Cry3Bb.11236B-21774 EG11237 May 27, 1997 Cry3Bb.11237 B-21775 EG11238 May 27, 1997Cry3Bb.11238 B-21776 EG11239 May 27, 1997 Cry3Bb.11239 B-21777 EG11241May 27, 1997 Cry3Bb.11241 B-21778 EG11242 May 27, 1997 Cry3Bb.11242B-21779

[0057] Also disclosed are methods of controlling or eradicating aninsect population from an environment. Such methods generally comprisecontacting the insect population to be controlled or eradicated with aninsecticidally-effective amount of a Cry3* crystal protein composition.Preferred Cry3* compositions include Cry3A*, Cry3B*, and Cry3C*polypeptide compositions, with Cry3B* compositions being particularlypreferred. Examples of such polypeptides include proteins selected fromthe group consisting of Cry3Bb-60, Cry3Bb.11221, Cry3Bb.11222,Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227,Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232,Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237,Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11032,Cry3Bb.11035, Cry3Bb.11036, Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051,Cry3Bb.11057, Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083,Cry3Bb.11084, Cry3Bb.11095, and Cry3Bb.11098.

[0058] In preferred embodiments, these Cry3Bb* crystal proteincompositions comprise the amino acid sequence of any of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102 or SEQ ID NO: 108.

[0059] 2.1 Methods for Producing Modified Cry* Proteins

[0060] The modified Cry* polypeptides of the present invention arepreparable by a process which generally involves the steps of obtaininga nucleic acid sequence encoding a Cry* polypeptide; analyzing thestructure of the polypeptide to identify particular “target” sites formutagenesis of the underlying gene sequence; introducing one or moremutations into the nucleic acid sequence to produce a change in one ormore amino acid residues in the encoded polypeptide sequence; andexpressing in a transformed host cell the mutagenized nucleic acidsequence under conditions effective to obtain the modified Cry* proteinencoded by the cry* gene.

[0061] Means for obtaining the crystal structures of the polypeptides ofthe invention are well-known. Exemplary high resolution crystalstructure solution sets are given in Section 9.0 of the disclosure, andinclude the crystal structure of both the Cry3A and Cry3B polypeptidesdisclosed herein. The information provided in Section 9.0 permits theanalyses disclosed in each of the methods herein which rely on the 3Dcrystal structure information for targeting mutagenesis of thepolypeptides to particular regions of the primary amino acid sequencesof the δ-endotoxins to obtain mutants with increased insecticidalactivity or enhanced insecticidal specificity.

[0062] A first method for producing a modified B. thuringiensis Cry3Bbδ-endotoxin having improved insecticidal activity or specificitydisclosed herein generally involves obtaining a high-resolution 3Dcrystal structure of the endotoxin, locating in the crystal structureone or more regions of bound water wherein the bound water forms acontiguous hydrated surfaces separated by no more than about 16 Å;increasing the number of water molecules in this surface by increasingthe hydrophobicity of one or more amino acids of the protein in theregion; and obtaining the modified δ-endotoxin so produced. Exemplaryδ-endotoxins include Cry3Bb.11032, Cry3Bb.11227, Cry3Bb.11241,Cry3Bb.11051, Cry3Bb.11242, and Cry3Bb.11098.

[0063] A second method for producing a modified B. thuringiensis Cry3Bbδ-endotoxin having improved insecticidal activity comprises identifyinga loop region in a δ-endotoxin; modifying one or more amino acids in theloop to increase the hydrophobicity of the amino acids; and obtainingthe modified δ-endotoxin so produced. Preferred δ-endotoxinproduced bythis method include Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11228,Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11233, Cry3Bb.11236,Cry3Bb.11237, Cry3Bb.11238, and Cry3Bb.11239.

[0064] A method for increasing the mobility of channel forming helicesof a B. thuringiensis Cry3B δ-endotoxin is also provided by the presentinvention. The method generally comprises disrupting one or morehydrogen bonds formed between a first amino acid of one or more of thechannel forming helices and a second amino acid of the δ-endotoxin. Thehydrogen bonds may be formed inter- or intramolecularly, and thedisrupting may consist of replacing a first or second amino acid with athird amino acid whose spatial distance is greater than about 3 Å, orwhose spatial orientation bond angle is not equal to 180±60 degreesrelative to the hydrogen bonding site of the first or second amino acid.δ-endotoxins produced by this method and disclosed herein includeCry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225, Cry3Bb.11226,Cry3Bb.11227, Cry3Bb.11231, Cry3Bb.11241, and Cry3Bb.11242, andCry3Bb.11098.

[0065] Also disclosed is a method of increasing the flexibility of aloop region in a channel forming domain of a B. thuringiensis Cry3Bbδ-endotoxin. This method comprises obtaining a crystal structure of aCry3Bb δ-endotoxin having one or more loop regions; identifying theamino acids comprising the loop region; and altering one or more of theamino acids to reduce steric hindrance in the loop region, wherein thealtering increases flexibility of the loop region in the δ-endotoxin.Examples of δ-endotoxins produced using this method includeCry3Bb.11032, Cry3Bb.11051, Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230,Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11236, Cry3Bb.11237,Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11227, Cry3Bb.11234, Cry3Bb.11241,Cry3Bb.11242, Cry3Bb.11036, and Cry3Bb.11098.

[0066] Another aspect of the invention is a method for increasing theactivity of a δ-endotoxin, comprising reducing or eliminating binding ofthe δ-endotoxin to a carbohydrate in a target insect gut. Theeliminating or reducing may be accomplished by removal of one or more αhelices of domain 1 of the δ-endotoxin, for example, by removal of αhelices α1, α2a/b, and α3. An exemplary δ-endotoxin produced using themethod is Cry3Bb.60.

[0067] Alternatively, the reducing or eliminating may be accomplished byreplacing one or more amino acids within loop β1,α8, with one or moreamino acids having increased hydrophobicity. Such a method gives rise toδ-endotoxins such as Cry3Bb.11228, Cry3Bb.11230, Cry3B.11231,Cry3Bb.11237, and Cry3Bb.11098, which are described in detail, herein.

[0068] Alternatively, the reducing or eliminating is accomplished byreplacing one or more specific amino acids, with any other amino acid.Such replacements are described in Table 2, and in the examples herein.One example is the δ-endotoxin designated herein as Cry3Bb.11221.

[0069] A method of identifying a region of a Cry3Bb δ-endotoxin fortargeted mutagenesis comprising: obtaining a crystal structure of theδ-endotoxin; identifying from the crystal structure one or moresurface-exposed amino acids in the protein; randomly substituting one ormore of the surface-exposed amino acids to obtain a plurality of mutatedpolypeptides, wherein at least 50% of the mutated polypeptides havediminished insecticidal activity; and identifying from the plurality ofmutated polypeptides one or more regions of the Cry3Bb δ-endotoxin fortargeted mutagenesis. The method may further comprise determining theamino acid sequences of a plurality of mutated polypeptides havingdiminished activity, and identifying one or more amino acid residuesrequired for insecticidal activity.

[0070] In another embodiment, the invention provides a process forproducing a Cry3Bb δ-endotoxin having improved insecticidal activity.The process generally involves the steps of obtaining a high-resolutioncrystal structure of the protein; determining the electrostatic surfacedistribution of the protein; identifying one or more regions of highelectrostatic diversity; modifying the electrostatic diversity of theregion by altering one or more amino acids in the region; and obtaininga Cry3Bb δ-endotoxin which has improved insecticidal activity. In oneembodiment, the electrostatic diversity may be decreased relative to theelectrostatic diversity of a native Cry3Bb δ-endotoxin. Exemplaryδ-endotoxins with decreased electrostatic diversity includeCry3Bb.11227, Cry3Bb.11241, and Cry3Bb.11242. Alternatively, theelectrostatic diversity may be increased relative to the electrostaticdiversity of a native Cry3Bb δ-endotoxin. An exemplary δ-endotoxin withincreased electrostatic diversity is Cry3Bb.11234.

[0071] Furthermore, the invention also provides a method of producing aCry3Bb δ-endotoxin having improved insecticidal activity which involvesobtaining a high-resolution crystal structure; identifying the presenceof one or more metal binding sites in the protein; altering one or moreamino acids in the binding site; and obtaining an altered protein,wherein the protein has improved insecticidal activity. The altering mayinvolve the elimination of one or more metal binding sites. Exemplaryδ-endotoxin include Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225, andCry3Bb.11226.

[0072] A further aspect of the invention involves a method ofidentifying a B. thuringiensis Cry3Bb δ-endotoxin having improvedchannel activity. This method in an overall sense involves obtaining aCry3Bb δ-endotoxin suspected of having improved channel activity; anddetermining one or more of the following characteristics in theδ-endotoxin, and comparing such characteristics to those obtained forthe wild-type unmodified δ-endotoxin; (1) the rate of channel formation,(2) the rate of growth of channel conductance or (3) the duration ofopen channel state. From this comparison, one may then select aδ-endotoxin which has an increased rate of channel formation compared tothe wildtype δ-endotoxin. Examples of Cry3Bb δ-endotoxins prepared bythis method include Cry3Bb.60, Cry3Bb.11035, Cry3Bb.11048, Cry3Bb.11032,Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11226, Cry3Bb.11221, Cry3Bb.11242,Cry3Bb.11230, and Cry3Bb.11098.

[0073] Also provided is a method for producing a modified Cry3Bbδ-endotoxin, having improved insecticidal activity which involvesaltering one or more non-surface amino acids located at or near thepoint of greatest convergence of two or more loop regions of the Cry3Bbδ-endotoxin, such that the altering decreases the mobility of one ormore of the loop regions. The mobility may conveniently be determined bycomparing the thermal denaturation of the modified protein to awild-type Cry3Bb δ-endotoxin. An exemplary crystal protein produced bythis method is Cry3Bb.11095.

[0074] A further aspect of the invention involves a method for preparinga modified Cry3Bb δ-endotoxin, having improved insecticidal activitycomprising modifying one or more amino acids in the loop to increase thehydrophobicity of said amino acids; and altering one or more of saidamino acids to reduce steric hindrance in the loop region, wherein thealtering increases flexibility of the loop region in the endotoxin.Exemplary Cry3Bb δ-endotoxins produced is selected from the groupconsisting of Cry3Bb.11057, Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082,Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11231, Cry3Bb.11235, andCry3Bb.11098.

[0075] The invention also provides a method of improving theinsecticidal activity of a B. thuringiensis Cry3Bb δ-endotoxin, whichgenerally comprises inserting one or more protease sensitive sites intoone or more loop regions of domain 1 of the δ-endotoxin. Preferably, theloop region is α3,4, and an exemplary δ-endotoxin so produced isCry3Bb.11221.

[0076] 2.2 Polypeptide Compositions

[0077] The crystal proteins so produced by each of the methods describedherein also represent important aspects of the invention. Such crystalproteins preferably include a protein or peptide selected from the groupconsisting of Cry3Bb-60, Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223,Cry3Bb.11224, Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228,Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233,Cry3Bb.11234, Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238,Cry3Bb.11239, Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035,Cry3Bb.11036, Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057,Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084,Cry3Bb.11095, and Cry3Bb.11098.

[0078] In preferred embodiments, the protein comprises a contiguousamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, and SEQ ID NO:108.

[0079] Highly preferred are those crystal proteins which are encoded bythe nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101; or SEQ ID NO:107, or anucleic acid sequence which hybridizes to the nucleic acid sequence ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO: 11, SEQ ID NO: 13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:99, SEQ ID NO:101, or SEQ ID NO:107 under conditions of moderatestringency.

[0080] Amino acid, peptide and protein sequences within the scope of thepresent invention include, and are not limited to the sequences setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22 SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70. SEQ ID NO:100, SEQ ID NO:102, and SEQ ID NO:108, and alterationsin the amino acid sequences including alterations, deletions, mutations,and homologs.

[0081] Compositions which comprise from about 0.5% to about 99% byweight of the crystal protein, or more preferably from about 5% to about75%, or from about 25% to about 50% by weight of the crystal protein areprovided herein. Such compositions may readily be prepared usingtechniques of protein production and purification well-known to those ofskill, and the methods disclosed herein. Such a process for preparing aCry3Bb* crystal protein generally involves the steps of culturing a hostcell which expresses the Cry3Bb* protein (such as a B. thuringiensisEG11221, EG11222, EG11223, EG11224, EG11225, EG11226, EG11227, EG11228,EG11229, EG11230, EG11231, EG11232, EG11233, EG11234, EG11235, EG11236,EG11237, EG11238, EG11239, EG11241, EG11242, EG11032, EG11035, EG11036,EG11046, EG11048, EG11051, EG11057, EG11058, EG11081, EG11082, EG11083,EG11084, EG11095, or EG11098 cell) under conditions effective to producethe crystal protein, and then obtaining the crystal protein so produced.

[0082] The protein may be present within intact cells, and as such, nosubsequent protein isolation or purification steps may be required.Alternatively, the cells may be broken, sonicated, lysed, disrupted, orplasmolyzed to free the crystal protein(s) from the remaining celldebris. In such cases, one may desire to isolate, concentrate, orfurther purify the resulting crystals containing the proteins prior touse, such as, for example, in the formulation of insecticidalcompositions. The composition may ultimately be purified to consistalmost entirely of the pure protein, or alternatively, be purified orisolated to a degree such that the composition comprises the crystalprotein(s) in an amount of from between about 0.5% and about 99% byweight, or in an amount of from between about 5% and about 95% byweight, or in an amount of from between about 15% and about 85% byweight, or in an amount of from between about 25% and about 75% byweight, or in an amount of from between about 40% and about 60% byweight etc.

[0083] 2.3 Recombinant Vectors Expressing Cry3* Genes

[0084] One important embodiment of the invention is a recombinant vectorwhich comprises a nucleic acid segment encoding one or more of the novelB. thuringiensis crystal proteins disclosed herein. Such a vector may betransferred to and replicated in a prokaryotic or eukaryotic host, withbacterial cells being particularly preferred as prokaryotic hosts, andplant cells being particularly preferred as eukaryotic hosts.

[0085] In preferred embodiments, the recombinant vector comprises anucleic acid segment encoding the amino acid sequence of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, or SEQ ID NO:108. Highly preferred nucleic acid segments arethose which have the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107.

[0086] Another important embodiment of the invention is a transformedhost cell which expresses one or more of these recombinant vectors. Thehost cell may be either prokaryotic or eukaryotic, and particularlypreferred host cells are those which express the nucleic acid segment(s)comprising the recombinant vector which encode one or more B.thuringiensis crystal protein comprising modified amino acid sequencesin one or more loop regions of domain 1, or between α helix 7 of domain1 and β strand 1 of domain 2. Bacterial cells are particularly preferredas prokaryotic hosts, and plant cells are particularly preferred aseukaryotic hosts

[0087] In an important embodiment, the invention discloses and claims ahost cell wherein the modified amino acid sequences comprise one or moreloop regions between α helices 1 and 2, α helices 2 and 3, α helices 3and 4, α helices 4 and 5, α helices 5 and 6 or α helices 6 and 7 ofdomain 1, or between α helix 7 of domain 1 and β strand 1 of domain 2. Aparticularly preferred host cell is one that comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID NO:108, and morepreferably, one that comprises the nucleic acid sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ IDNO:101, or SEQ ID NO:107.

[0088] Bacterial host cells transformed with a nucleic acid segmentencoding a modified Cry3Bb crystal protein according to the presentinvention are disclosed and claimed herein, and in particular, a B.thuringiensis cell having designation EG11221, EG11222, EG11223,EG11224, EG11225, EG11226, EG11227, EG11228, EG11229, EG11230, EG11231,EG11232, EG11233, EG11234, EG11235, EG11236, EG11237, EG11238, EG11239,EG11241, EG11242, EG11032, EG11035, EG11036, EG11046, EG11048, EG11051,EG11057, EG11058, EG11081, EG11082, EG11083, EG11084, EG11095, orEG11098.

[0089] In another embodiment, the invention encompasses a method ofusing a nucleic acid segment of the present invention that encodes acry3Bb* gene. The method generally comprises the steps of: (a) preparinga recombinant vector in which the cry3Bb* gene is positioned under thecontrol of a promoter; (b) introducing the recombinant vector into ahost cell; (c) culturing the host cell under conditions effective toallow expression of the Cry3Bb* crystal protein encoded by said cry3Bb*gene; and (d) obtaining the expressed Cry3Bb* crystal protein orpeptide.

[0090] A wide variety of ways are available for introducing a B.thuringiensis gene expressing a toxin into the microorganism host underconditions which allow for stable maintenance and expression of thegene. One can provide for DNA constructs which include thetranscriptional and translational regulatory signals for expression ofthe toxin gene, the toxin gene under their regulatory control and a DNAsequence homologous with a sequence in the host organism, wherebyintegration will occur, and/or a replication system which is functionalin the host, whereby integration or stable maintenance will occur.

[0091] The transcriptional initiation signals will include a promoterand a transcriptional initiation start site. In some instances, it maybe desirable to provide for regulative expression of the toxin, whereexpression of the toxin will only occur after release into theenvironment. This can be achieved with operators or a region binding toan activator or enhancers, which are capable of induction upon a changein the physical or chemical environment of the microorganisms. Forexample, a temperature sensitive regulatory region may be employed,where the organisms may be grown up in the laboratory without expressionof a toxin, but upon release into the environment, expression wouldbegin. Other techniques may employ a specific nutrient medium in thelaboratory, which inhibits the expression of the toxin, where thenutrient medium in the environment would allow for expression of thetoxin. For translational initiation, a ribosomal binding site and aninitiation codon will be present.

[0092] Various manipulations may be employed for enhancing theexpression of the messenger RNA, particularly by using an activepromoter, as well as by employing sequences, which enhance the stabilityof the messenger RNA. The transcriptional and translational terminationregion will involve stop codon(s), a terminator region, and optionally,a polyadenylation signal. A hydrophobic “leader” sequence may beemployed at the amino terminus of the translated polypeptide sequence inorder to promote secretion of the protein across the inner membrane.

[0093] In the direction of transcription, namely in the 5′ to 3′direction of the coding or sense sequence, the construct will involvethe transcriptional regulatory region, if any, and the promoter, wherethe regulatory region may be either 5′ or 3′ of the promoter, theribosomal binding site, the initiation codon, the structural gene havingan open reading frame in phase with the initiation codon, the stopcodon(s), the polyadenylation signal sequence, if any, and theterminator region. This sequence as a double strand may be used byitself for transformation of a microorganism host, but will usually beincluded with a DNA sequence involving a marker, where the second DNAsequence may be joined to the toxin expression construct duringintroduction of the DNA into the host.

[0094] By a marker is intended a structural gene which provides forselection of those hosts which have been modified or transformed. Themarker will normally provide for selective advantage, for example,providing for biocide resistance, e.g., resistance to antibiotics orheavy metals; complementation, so as to provide prototropy to anauxotrophic host, or the like. Preferably, complementation is employed,so that the modified host may not only be selected, but may also becompetitive in the field. One or more markers may be employed in thedevelopment of the constructs, as well as for modifying the host. Theorganisms may be further modified by providing for a competitiveadvantage against other wild-type microorganisms in the field. Forexample, genes expressing metal chelating agents, e.g., siderophores,may be introduced into the host along with the structural geneexpressing the toxin. In this manner, the enhanced expression of asiderophore may provide for a competitive advantage for thetoxin-producing host, so that it may effectively compete with thewild-type microorganisms and stably occupy a niche in the environment.

[0095] Where no functional replication system is present, the constructwill also include a sequence of at least 50 basepairs (bp), preferablyat least about 100 bp, more preferably at least about 1000 bp, andusually not more than about 2000 bp of a sequence homologous with asequence in the host. In this way, the probability of legitimaterecombination is enhanced, so that the gene will be integrated into thehost and stably maintained by the host. Desirably, the toxin gene willbe in close proximity to the gene providing for complementation as wellas the gene providing for the competitive advantage. Therefore, in theevent that a toxin gene is lost, the resulting organism will be likelyto also lost the complementing gene and/or the gene providing for thecompetitive advantage, so that it will be unable to compete in theenvironment with the gene retaining the intact construct.

[0096] A large number of transcriptional regulatory regions areavailable from a wide variety of microorganism hosts, such as bacteria,bacteriophage, cyanobacteria, algae, fungi, and the like. Varioustranscriptional regulatory regions include the regions associated withthe trp gene, lac gene, gal gene, the λ_(L) and λ_(R) promoters, the tacpromoter, the naturally-occurring promoters associated with theδ-endotoxin gene, where functional in the host. See for example, U.S.Pat. Nos. 4,332,898; 4,342,832; and 4,356,270 (each of which isspecifically incorporated herein by reference). The termination regionmay be the termination region normally associated with thetranscriptional initiation region or a different transcriptionalinitiation region, so long as the two regions are compatible andfunctional in the host.

[0097] Where stable episomal maintenance or integration is desired, aplasmid will be employed which has a replication system which isfunctional in the host. The replication system may be derived from thechromosome, an episomal element normally present in the host or adifferent host, or a replication system from a virus which is stable inthe host. A large number of plasmids are available, such as pBR322,pACYC184, RSF1010, pR01614. and the like. See for example, Olson et al.(1982); Bagdasarian et al. (1981), Baum et al., 1990, and U.S. Pat. Nos.4,356,270; 4,362,817; 4,371,625, and 5,441,884, each incorporatedspecifically herein by reference.

[0098] The B. thuringiensis gene can be introduced between thetranscriptional and translational initiation region and thetranscriptional and translational termination region, so as to be underthe regulatory control of the initiation region. This construct will beincluded in a plasmid, which will include at least one replicationsystem, but may include more than one, where one replication system isemployed for cloning during the development of the plasmid and thesecond replication system is necessary for functioning in the ultimatehost. In addition, one or more markers may be present, which have beendescribed previously. Where integration is desired, the plasmid willdesirably include a sequence homologous with the host genome.

[0099] The transformants can be isolated in accordance with conventionalways, usually employing a selection technique, which allows forselection of the desired organism as against unmodified organisms ortransferring organisms, when present. The transformants then can betested for pesticidal activity. If desired, unwanted or ancillary DNAsequences may be selectively removed from the recombinant bacterium byemploying site-specific recombination systems, such as those describedin U.S. Pat. No. 5,441,884 (specifically incorporated herein byreference).

[0100] 2.4 Cry3 DNA Segments

[0101] A B. thuringiensis cry3* gene encoding a crystal protein havingone or more mutations in one or more regions of the peptide representsan important aspect of the invention. Preferably, the cry3* gene encodesan amino acid sequence in which one or more amino acid residues havebeen changed based on the methods disclosed herein, and particularlythose changes which have been made for the purpose of altering theinsecticidal activity or specificity of the crystal protein.

[0102] In accordance with the present invention, nucleic acid sequencesinclude and are not limited to DNA, including and not limited to cDNAand genomic DNA, genes; RNA, including and not limited to mRNA and tRNA;antisense sequences, nucleosides, and suitable nucleic acid sequencessuch as those set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107, andalterations in the nucleic acid sequences including alterations,deletions, mutations, and homologs capable of expressing the B.thuringiensis modified toxins of the present invention.

[0103] As such the present invention also concerns DNA segments, thatare free from total genomic DNA and that encode the novelsynthetically-modified crystal proteins disclosed herein. DNA segmentsencoding these peptide species may prove to encode proteins,polypeptides, subunits, functional domains, and the like of crystalprotein-related or other non-related gene products. In addition theseDNA segments may be synthesized entirely in vitro using methods that arewell-known to those of skill in the art.

[0104] As used herein, the term “DNA segment” refers to a DNA moleculethat has been isolated free of total genomic DNA of a particularspecies. Therefore, a DNA segment encoding a crystal protein or peptiderefers to a DNA segment that contains crystal protein coding sequencesyet is isolated away from, or purified free from, total genomic DNA ofthe species from which the DNA segment is obtained, which in the instantcase is the genome of the Gram-positive bacterial genus, Bacillus, andin particular, the species of Bacillus known as B. thuringiensis.Included within the term “DNA segment”, are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phagemids, phage, viruses, and the like.

[0105] Similarly, a DNA segment comprising an isolated or purifiedcrystal protein-encoding gene refers to a DNA segment which may includein addition to peptide encoding sequences, certain other elements suchas, regulatory sequences, isolated substantially away from othernaturally occurring genes or protein-encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein-, polypeptide- or peptide-encoding unit. As will be understoodby those in the art, this functional term includes both genomicsequences, operon sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides orpeptides.

[0106] “Isolated substantially away from other coding sequences” meansthat the gene of interest, in this case, a gene encoding a bacterialcrystal protein, forms the significant part of the coding region of theDNA segment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or operon coding regions. Of course, this refersto the DNA segment as originally isolated, and does not exclude genes,recombinant genes, synthetic linkers, or coding regions later added tothe segment by the hand of man.

[0107] Particularly preferred DNA sequences are those encodingCry3Bb.60, Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224,Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228, Cry3Bb.11229,Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234,Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239,Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036,Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057, Cry3Bb.11058,Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11095 andCry3Bb.11098 crystal proteins, and in particular cry3Bb* genes such ascry3Bb.60, cry3Bb.11221, cry3Bb.11222, cry3Bb.11223, cry3Bb.11224,cry3Bb.11225, cry3Bb.11226, cry3Bb. 11227, cry3Bb.11228, cry3Bb.11229,cry3Bb.11230, cry3Bb.11231, cry3Bb.11232, cry3Bb.11233, cry3Bb.11234,cry3Bb.11235, cry3Bb.11236, cry3Bb.11237, cry3Bb.11238, cry3Bb.11239,cry3Bb.11241, cry3Bb.11242, cry3Bb.110322 cry3Bb.11035, cry3Bb.11036,cry3Bb.11046, cry3Bb.11048, cry3Bb.11051, cry3Bb.11057, cry3Bb.11058,cry3Bb.11081, cry3Bb.11082, cry3Bb.11083, cry3Bb.11084, cry3Bb.11095 andcry3Bb.11098. In particular embodiments, the invention concerns isolatedDNA segments and recombinant vectors incorporating DNA sequences thatencode a Cry peptide species that includes within its amino acidsequence an amino acid sequence essentially as set forth in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, or SEQ ID NO:108.

[0108] The term “a sequence essentially as set forth in SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, or SEQ ID NO:108” means that the sequence substantiallycorresponds to a portion of the sequence of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQID NO:108, and has relatively few amino acids that are not identical to,or a biologically functional equivalent of, the amino acids of any ofthese sequences. The term “biologically functional equivalent” is wellunderstood in the art and is further defined in detail herein (e.g., seeIllustrative Embodiments).

[0109] Accordingly, sequences that have between about 70% and about 75%or between about 75% and about 80%, or more preferably between about 81%and about 90%, or even more preferably between about 91% or 92% or 93%and about 97% or 98% or 99% amino acid sequence identity or functionalequivalence to the amino acids of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102 or SEQ ID NO:108 willbe sequences that are “essentially as set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, or SEQ ID NO:108.”

[0110] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences. and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding regionor may include various internal sequences, i.e., introns, which areknown to occur within genes.

[0111] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

[0112] For example, nucleic acid fragments may be prepared that includea short contiguous stretch encoding the peptide sequence disclosed inSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ IDNO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ IDNO:100, SEQ ID NO:102, or SEQ ID NO:108, or that are identical to orcomplementary to DNA sequences which encode the peptide disclosed in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ IDNO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68. SEQ ID NO:70, SEQ IDNO:100, SEQ ID NO:102, or SEQ ID NO:108, and particularly the DNAsegments disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107.

[0113] Highly preferred nucleic acid segments of the present inventioncomprise one or more cry genes of the invention, or a portion of one ormore cry genes of the invention. For certain application, relativelysmall contiguous nucleic acid sequences are preferable, such as thosewhich are about 14 or 15 or 16 or 17 or 18 or 19, or 20, or 30-50,51-80, 81-100 or so nucleotides in length. Alternatively, in someembodiments, and particularly those involving preparation of recombinantvectors, transformation of suitable host cells, and preparation oftransgenic plant cell, longer nucleic acid segments are preferred,particularly those that include the entire coding region of one or morecry genes. As such, the preferred segments may include those that are upto about 20,000 or so nucleotides in length, or alternatively, shortersequences such as those about 19,000, about 18,000, about 17,000, about16,000, about 15,000, about 14,000, about 13,000, about 12,000, 11,000,about 10,000, about 9,000, about 8,000, about 7,000, about 6,000, about5,000, about 4,500, about 4,000, about 3,500, about 3,000, about 2,500,about 2,000, about 1,500, about 1,000, about 500, or about 200 or sobase pairs in length. Of course, these numbers are not intended to beexclusionary of all possible intermediate lengths in the range of fromabout 20,000 to about 15 nucleotides, as all of these intermediatelengths are also contemplated to be useful, and fall within the scope ofthe present invention. It will be readily understood that “intermediatelengths”, in these contexts, means any length between the quoted ranges,such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, 24, 25, 26, 27,28, 29, etc.; 30, 31, 32, 33, 34, 35, 36 . . . etc.; 40, 41, 42, 43, 44. . . etc., 50, 51, 52, 53 . . . etc.; 60, 61, 62, 63 . . . etc., 70,80, 90, 100, 110, 120, 130 . . . etc.; 200, 210, 220, 230, 240, 250 . .. etc.; including all integers in the entire range from about 14 toabout 10,000, including those integers in the ranges 200-500; 500-1,000;1,000-2,000; 2,000-3,000; 3,000-5,000 and the like.

[0114] In a preferred embodiment, the nucleic acid segments comprise asequence of from about 1800 to about 18,000 base pair in length, andcomprise one or more genes which encode a modified Cry3* polypeptidedisclosed herein which has increased activity against Coleopteran insectpests.

[0115] It will also be understood that this invention is not limited tothe particular nucleic acid sequences which encode peptides of thepresent invention, or which encode the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100,SEQ ID NO:102, or SEQ ID NO:108, including the DNA sequences which areparticularly disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107.Recombinant vectors and isolated DNA segments may therefore variouslyinclude the peptide-coding regions themselves, coding regions bearingselected alterations or modifications in the basic coding region, orthey may encode larger polypeptides that nevertheless include thesepeptide-coding regions or may encode biologically functional equivalentproteins or peptides that have variant amino acids sequences.

[0116] The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level

[0117] If desired, one may also prepare fusion proteins and peptides,e.g., where the peptide-coding regions are aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins that may be purified by affinity chromatography and enzymelabel coding regions, respectively).

[0118] Recombinant vectors form further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the DNA segment, whether encodinga full length protein or smaller peptide, is positioned under thecontrol of a promoter. The promoter may be in the form of the promoterthat is naturally associated with a gene encoding peptides of thepresent invention, as may be obtained by isolating the 5′ non-codingsequences located upstream of the coding segment or exon, for example,using recombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

[0119] 2.5 Vectors, Host Cells, and Protein Expression

[0120] In other embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a DNA segment encoding a crystalprotein or peptide in its natural environment. Such promoters mayinclude promoters normally associated with other genes, and/or promotersisolated from any bacterial, viral, eukaryotic, or plant cell.Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the cell type, organism, oreven animal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al., 1989.The promoters employed may be constitutive, or inducible, and can beused under the appropriate conditions to direct high level expression ofthe introduced DNA segment, such as is advantageous in the large-scaleproduction of recombinant proteins or peptides. Appropriate promotersystems contemplated for use in high-level expression include, but arenot limited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

[0121] In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8, 9, 10, or 11 or soamino acids, and up to and including those of about 30, 40, or 50 or soamino acids in length, or more preferably, from about 8 to about 30amino acids in length, or even more preferably, from about 8 to about 20amino acids in length are contemplated to be particularly useful. Suchpeptide epitopes may be amino acid sequences which comprise contiguousamino acid sequence from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID NO:108.

[0122] 2.6 Transformed Host Cells and Transgenic Plants

[0123] In one embodiment, the invention provides a transgenic planthaving incorporated into its genome a transgene that encodes acontiguous amino acid sequence selected from the group consisting of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ IDNO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ IDNO:100, SEQ ID NO:102, and SEQ ID NO:108.

[0124] A further aspect of the invention is a transgenic plant havingincorporated into its genome a cry3Bb* transgene, provided the transgenecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:99, SEQ ID NO:101, and SEQ ID NO:107. Also disclosed and claimed areprogeny of such a transgenic plant, as well as its seed, progeny fromsuch seeds, and seeds arising from the second and subsequent generationplants derived from such a transgenic plant.

[0125] The invention also discloses and claims host cells, both native,and genetically engineered, which express the novel cry3Bb* genes toproduce Cry3Bb* polypeptides. Preferred examples of bacterial host cellsinclude B. thuringiensis EG11221, EG11222, EG11223, EG11224, EG11225,EG11226, EG11227, EG11228, EG11229, EG11230, EG11231, EG11232, EG11233,EG11234, EG11235, EG11236, EG11237, EG11238, EG11239, EG11241, EG11242,EG11032, EG11035, EG11036, EG11046, EG11048, EG11051, EG11057, EG11058,EG11081, EG11082, EG11083, EG11084, EG11095, and EG11098.

[0126] Methods of using such cells to produce Cry3* crystal proteins arealso disclosed. Such methods generally involve culturing the host cell(such as B. thuringiensis EG 11221, EG11222, EG11223, EG11224, EG11225,EG11226, EG11227, EG11228, EG11229, EG11230, EG11231, EG11232, EG11233,EG11234, EG11235, EG11236, EG11237. EG11238, EG11239, EG11241, EG11242,EG11032, EG11035, EG11036, EG11046, EG11048, EG11051, EG11057, EG11058,EG11081, EG11082, EG11083, EG11084, or EG11095, or EG11098) underconditions effective to produce a Cry3* crystal protein, and obtainingthe Cry3* crystal protein from said cell.

[0127] In yet another aspect, the present invention provides methods forproducing a transgenic plant which expresses a nucleic acid segmentencoding the novel recombinant crystal proteins of the presentinvention. The process of producing transgenic plants is well-known inthe art. In general, the method comprises transforming a suitable hostcell with one or more DNA segments which contain one or more promotersoperatively linked to a coding region that encodes one or more of thedisclosed B. thuringiensis crystal proteins. Such a coding region isgenerally operatively linked to a transcription-terminating region,whereby the promoter is capable of driving the transcription of thecoding region in the cell, and hence providing the cell the ability toproduce the recombinant protein in vivo. Alternatively, in instanceswhere it is desirable to control, regulate, or decrease the amount of aparticular recombinant crystal protein expressed in a particulartransgenic cell, the invention also provides for the expression ofcrystal protein antisense mRNA. The use of antisense mRNA as a means ofcontrolling or decreasing the amount of a given protein of interest in acell is well-known in the art.

[0128] Another aspect of the invention comprises a transgenic plantwhich express a gene or gene segment encoding one or more of the novelpolypeptide compositions disclosed herein. As used herein, the term“transgenic plant” is intended to refer to a plant that has incorporatedDNA sequences, including but not limited to genes which are perhaps notnormally present, DNA sequences not normally transcribed into RNA ortranslated into a protein (“expressed”), or any other genes or DNAsequences which one desires to introduce into the non-transformed plant,such as genes which may normally be present in the non-transformed plantbut which one desires to either genetically engineer or to have alteredexpression.

[0129] It is contemplated that in some instances the genome of atransgenic plant of the present invention will have been augmentedthrough the stable introduction of one or more Cry3Bb*-encodingtransgenes, either native, synthetically modified, or mutated. In someinstances, more than one transgene will be incorporated into the genomeof the transformed host plant cell. Such is the case when more than onecrystal protein-encoding DNA segment is incorporated into the genome ofsuch a plant. In certain situations, it may be desirable to have one,two, three, four, or even more B. thuringiensis crystal proteins (eithernative or recombinantly-engineered) incorporated and stably expressed inthe transformed transgenic plant.

[0130] A preferred gene which may be introduced includes, for example, acrystal protein-encoding a DNA sequence from bacterial origin, andparticularly one or more of those described herein which are obtainedfrom Bacillus spp. Highly preferred nucleic acid sequences are thoseobtained from B. thuringiensis, or any of those sequences which havebeen genetically engineered to decrease or increase the insecticidalactivity of the crystal protein in such a transformed host cell.

[0131] Means for transforming a plant cell and the preparation of atransgenic cell line are well-known in the art, and are discussedherein. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes)and DNA segments for use in transforming such cells will, of course,generally comprise either the operons, genes, or gene-derived sequencesof the present invention, either native, or synthetically-derived, andparticularly those encoding the disclosed crystal proteins. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even gene sequences which have positively- ornegatively-regulating activity upon the particular genes of interest asdesired. The DNA segment or gene may encode either a native or modifiedcrystal protein, which will be expressed in the resultant recombinantcells, and/or which will impart an improved phenotype to the regeneratedplant

[0132] Such transgenic plants may be desirable for increasing theinsecticidal resistance of a monocotyledonous or dicotyledonous plant,by incorporating into such a plant, a transgenic DNA segment encoding aCry3Bb* crystal protein which is toxic to coleopteran insects.Particularly preferred plants include grains such as corn, wheat, rye,rice, barley, and oats; legumes such as soybeans; tubers such aspotatoes; fiber crops such as flax and cotton; turf and pasture grasses;ornamental plants; shrubs; trees; vegetables, berries, citrus, fruits,cacti, succulents, and other commercially-important crops includinggarden and houseplants.

[0133] In a related aspect, the present invention also encompasses aseed produced by the transformed plant, a progeny from such seed, and aseed produced by the progeny of the original transgenic plant, producedin accordance with the above process. Such progeny and seeds will haveone or more crystal protein transgene(s) stably incorporated into itsgenome, and such progeny plants will inherit the traits afforded by theintroduction of a stable transgene in Mendelian fashion. All suchtransgenic plants having incorporated into their genome transgenic DNAsegments encoding one or more Cry3Bb* crystal proteins or polypeptidesare aspects of this invention. Particularly preferred transgenes for thepractice of the invention include nucleic acid segments comprising oneor more cry3Bb* gene(s).

[0134] 2.7 Biological Functional Equivalents

[0135] Modification and changes may be made in the structure of thepeptides of the present invention and DNA segments which encode them andstill obtain a functional molecule that encodes a protein or peptidewith desirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated crystal proteins are contemplated to be usefulfor increasing the insecticidal activity of the protein, andconsequently increasing the insecticidal activity and/or expression ofthe recombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 4. TABLE 4 Amino Acids Codons Alanine Ala A GCAGCC GCG GCU Cysteine Cys C UGC UGU Aspartic Acid Asp D GAC GAU GlutamicAcid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGGGGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG GUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0136] For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. It is thus contemplated by theinventors that various changes may be made in the peptide sequences ofthe disclosed compositions, or corresponding DNA sequences which encodesaid peptides without appreciable loss of their biological utility oractivity.

[0137] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

[0138] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte andDoolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0139] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0140] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, specifically incorporated herein by reference,states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the protein.

[0141] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0142] It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologicallyequivalent protein. In such changes, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those which arewithin ±1 are particularly preferred, and those within ±0.5 are evenmore particularly preferred.

[0143] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

[0144] The drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0145]FIG. 1. Schematic representation of the monomeric structure ofCry3Bb.

[0146]FIG. 2. Stereoscopic view of the monomeric structure of Cry3Bbwith associated water molecules (represented by dots).

[0147]FIG. 3A. Schematic representation of domain 1 of Cry3Bb

[0148]FIG. 3B. Diagram of the positions of the 7 helices that comprisedomain 1.

[0149]FIG. 4. Domain 1 of Cry3Bb is organized into seven α helicesillustrated in FIG. 3A (schematic representation) and FIG. 3B (schematicdiagram). The α helices and amino acids residues are shown.

[0150]FIG. 5A. Schematic representation of domain 2 of Cry3Bb.

[0151]FIG. 5B. Diagram of the positions of the 11 β strands that composethe 3 βsheets of domain 2.

[0152]FIG. 6. Domain 2 of Cry3Bb is a collection of three anti-parallelβ sheets illustrated in FIG. 5. The amino acids that define these sheetsis listed below (α8, amino aids 322-328, also is included in domain 2):

[0153]FIG. 7A. Schematic representation of domain 3 of Cry3Bb.

[0154]FIG. 7B. Diagram of the positions of the β strands that comprisedomain 3.

[0155]FIG. 8. Domain 3 (FIG. 7) is a loosely organized collection of βstrands and loops; no β sheets are present. The β stands contain theamino acids limited below:

[0156]FIG. 9A. A “side” view of the dimeric structure of Cry3Bb. Thehelical bundles of domains 1 can be seem in the middle of the molecule.

[0157]FIG. 9B. A “top” view of the dimeric structure of Cry3Bb. Thehelical bundles of domains 1 can be seem in the middle of the molecule.

[0158]FIG. 10. A graphic representation of the growth in conductancewith time of channels formed by Cry3A and Cry3Bb in planar lipidbilayers. Cry3A forms channels with higher conductances much morerapidly than Cry3Bb.

[0159]FIG. 11. A map of pEG1701 which contains the Cry3Bb gene with thecry1F terminator.

[0160]FIG. 12. The results of replicated 1-dose assays against SCRWlarvae of Cry3Bb proteins altered in the lB2,3 region.

[0161]FIG. 13. The results of replicated, 1-dose assays against SCRWlarvae of Cry3Bb proteins altered in the lB6, 7 region.

[0162]FIG. 14. The results of replicated, 1-dose screens against SCRWlarvae of Cry3Bb proteins altered in the lB10,11 region.

[0163]FIG. 15. Single channel recordings of channels formed byCry3Bb.11230 and WT Cry3Bb in planar lipid bilayers. Cry3Bb.11230 formschannels with well resolved open and closed states while Cry3Bb rarelydoes.

[0164]FIG. 16. Single channel recordings of channels formed by Cry3Bband Cry3Bb.60, a truzncated form of Cry3Bb. Cry3Bb.60 forms channelsmore quickly than Cry3Bb and, unlike Cry3Bb, produces channels with wellresolved open and closed states.

[0165]FIG. 17A. Sequence alignment of the amino acid sequence of Cry3A,Cry3B, and Cry3C.

[0166]FIG. 17B. Shown is a continuation of alignment of the amino acidsequence of Cry3A, Cry3B, and Cry3C shown in FIG. 17A.

[0167]FIG. 17C. Shown is a continuation of alignment of the amino acidsequence of Cry3A, Cry3B, and Cry3C shown in FIG. 17A.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0168] The invention defines new B. thuringiensis (Bt) insecticidalδ-endotoxin proteins and the biochemical and biophysical strategies usedto design the new proteins. Delta-endotoxins are a class of insecticdalproteins produced by B. thuringiensis that form cation-selectivechannels in planar lipid bilayers (English and Slatin, 1992). The newδ-endotoxins are based on the parent structure of thecoleopteran-active, δ-endotoxin Cry3Bb. Like other members of thecoleopteran-active class of δ-endotoxins, including Cry3A and Cry3B,Cry3Bb exhibits excellent insecticidal activity against the ColoradoPotato Beetle (Leptinotarsa decemlineata). However, unlike Cry3A andCry3B, Cry3Bb is also active against the southern corn rootworm or SCRW(Diabrotica undecimpunctata howardi Barber) and the western cornrootworm or WCRW (Diabrotica virgifera virgifera LeConte). The newinsecticidal proteins described herein were specifically designed toimprove the biological activity of the parent Cry3Bb protein. Inaddition, the design strategies themselves are novel inventions capableof being applied to and improving B. thuringiensis δ-endotoxins ingeneral. B. thuringiensis δ-endotoxins are also members of a largerclass of bacterial toxins that form ion channels (see English and Slatin1992, for a review). The inventors, therefore, believe that these designstrategies can also be applied to any biologically active,channel-forming protein to improve its biological properties.

[0169] The designed Cry3Bb proteins were engineered using one or more ofthe following strategies including (1) identification and alteration ofprotease-sensitive sites and proteolytic processing; (2) analysis andmanipulation of bound water; (3) manipulation of hydrogen bonds aroundmobile regions; (4) loop analysis and loop redesign around flexiblehelices; (5) loop design around β strands and β sheets; (6)identification and redesign of complex electrostatic surfaces; (7)identification and removal of metal binding sites; (8) alteration ofquaternary structure; (9) identification and design of structuralresidues; and (10) combinations of any and all sites defined bystrategies 1-9. These design strategies permit the identification andredesign of specific sites on Cry3Bb, ultimately creating new proteinswith improved insecticidal activities. These new proteins are designatedCry3Bb designed proteins and are named Cry3Bb followed by a period and asuffix (e.g., Cry3Bb.60, Cry3Bb.11231). The new proteins are listed inTable 2 along with the specific sites on the molecule that weremodified, the amino-acid sequence changes at those sites that improvebiological activity, the improved insecticidal activities and the designmethod used to identify that specific site.

[0170] 4.1 Some Advantages of the Invention

[0171] Mutagenesis studies with cry genes have failed to identify asignificant number of mutant crystal proteins which have improvedbroad-spectrum insecticidal activity, that is, with improved toxicitytowards a range of insect pest species. Since agricultural crops aretypically threatened by more than one insect pest species at any giventime, desirable mutant crystal proteins are preferably those thatexhibit improvements in toxicity towards multiple insect pest species.Previous failures to identify such mutants may be attributed to thechoice of sites targeted for mutagenesis. For example, with respect tothe related protein, Cry1C, sites within domain 2 and domain 3 have beenthe principal targets of mutagenesis efforts, primarily because thesedomains are believed to be important for receptor binding and indetermining insecticidal specificity (Aronson et al., 1995; Chen et al.1993; de Maagd et al., 1996; Lee et al., 1992; Lee et al., 1995; Lu etal., 1994; Smedley and Ellar, 1996; Smith and Ellar, 1994; Rajamohan etal., 1995; Rajamohan et al., 1996)

[0172] In contrast, the present inventors reasoned that the toxicity ofCry3 proteins, and specifically the toxicity of the Cry3Bb protein, maybe improved against a broader array of target pests by targeting regionsinvolved in ion channel function rather than regions of the moleculedirectly involved in receptor interactions, namely domains 2 and 3.Accordingly, the inventors opted to target regions within domain 1 ofCry3Bb for mutagenesis for the purpose of isolating Cry3Bb mutants withimproved broad spectrum toxicity. Indeed, in the present invention,Cry3Bb mutants are described that show improved toxicity towards severalcoleopteran pests.

[0173] At least one, and probably more than one, α helix of domain 1 isinvolved in the formation of ion channels and pores within the insectmidgut epithelium (Gazit and Shai, 1993; Gazit and Shai, 1995). Ratherthan target for mutagenesis the sequences encoding the α helices ofdomain 1 as others have (Wu and Aronson, 1992; Aronson et al., 1995;Chen et al., 1995), the present inventors opted to target exclusivelysequences encoding amino acid residues adjacent to or lying within thepredicted loop regions of Cry3Bb that separate these α helices. Aminoacid residues within these loop regions or amino acid residues cappingthe end of an α helix and lying adjacent to these loop regions mayaffect the spatial relationships among these α helices. Consequently,the substitution of these amino acid residues may result in subtlechanges in tertiary structure, or even quaternary structure, thatpositively impact the function of the ion channel. Amino acid residuesin the loop regions of domain 1 are exposed to the solvent and thus areavailable for various molecular interactions. Altering these amino acidscould result in greater stability of the protein by eliminating oroccluding protease-sensitive sites. Amino acid substitutions that changethe surface charge of domain 1 could alter ion channel efficiency oralter interactions with the brush border membrane or with other portionsof the toxin molecule, allowing binding or insertion to be moreeffective.

[0174] According to this invention, base substitutions are made in theunderlying cry3Bb nucleic acid residues in order to change particularcodons of the corresponding polypeptides, and particularly, in thoseloop regions between α-helices. The insecticidal activity of a crystalprotein ultimately dictates the level of crystal protein required foreffective insect control. The potency of an insecticidal protein shouldbe maximized as much as possible in order to provide for its economicand efficient utilization in the field. The increased potency of aninsecticidal protein in a bioinsecticide formulation would be expectedto improve the field performance of the bioinsecticide product.Alternatively, increased potency of an insecticidal protein in abioinsecticide formulation may promote use of reduced amounts ofbioinsecticide per unit area of treated crop, thereby allowing for morecost-effective use of the bioinsecticide product. When expressed inplanta, the production of crystal proteins with improved insecticidalactivity can be expected to improve plant resistance to susceptibleinsect pests.

[0175] 4.2 Methods for Culturing B. thuringiensis to Produce CrystalProteins

[0176] The B. thuringiensis strains described herein may be culturedusing standard known media and fermentation techniques. Upon completionof the fermentation cycle, the bacteria may be harvested by firstseparating the B. thuringiensis spores and crystals from thefermentation broth by means well known in the art. The recovered B.thuringiensis spores and crystals can be formulated into a wettablepowder, a liquid concentrate, granules or other formulations by theaddition of surfactants, dispersants, inert carriers and othercomponents to facilitate handling and application for particular targetpests. The formulation and application procedures are all well known inthe art.

[0177] 4.3 Recombinant Host Cells for Expression of Cry* Genes

[0178] The nucleotide sequences of the subject invention can beintroduced into a wide variety of microbial hosts. Expression of thetoxin gene results, directly or indirectly, in the intracellularproduction and maintenance of the pesticide. With suitable hosts, e.g.,Pseudomonas, the microbes can be applied to the sites of coleopteraninsects where they will proliferate and be ingested by the insects. Theresult is a control of the unwanted insects. Alternatively, the microbehosting the toxin gene can be treated under conditions that prolong theactivity of the toxin produced in the cell. The treated cell then can beapplied to the environment of target pest(s). The resulting productretains the toxicity of the B. thuringiensis toxin.

[0179] Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility or toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and Gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

[0180] Characteristics of particular interest in selecting a host cellfor purposes of production include ease of introducing the B.thuringiensis gene into the host, availability of expression systems,efficiency of expression, stability of the pesticide in the host, andthe presence of auxiliary genetic capabilities. Characteristics ofinterest for use as a pesticide micro-capsule include protectivequalities for the pesticide, such as thick cell walls, pigmentation, andintracellular packaging or formation of inclusion bodies; leaf affinity;lack of mammalian toxicity; attractiveness to pests for ingestion; easeof killing and fixing without damage to the toxin; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

[0181] Host organisms of particular interest include yeast, such asRhodotorula sp., Aureobasidium sp., Saccharomyces sp., andSporobolomyces sp.; phylloplane organisms such as Pseudomonas sp.,Erwinia sp. and Flavobacterium sp.; or such other organisms asEscherichia, Lactobacillus sp., Bacillus sp., Streptomyces sp., and thelike. Specific organisms include Pseudomonas aeruginosa, Pseudomonasfluorescens, Saccharomyces cerevisiae, B. thuringiensis, Escherichiacoli, B. subtilis, B. megaterium, B. cereus, Streptomyces lividans andthe like.

[0182] Treatment of the microbial cell, e. g., a microbe containing theB. thuringiensis toxin gene, can be by chemical or physical means, or bya combination of chemical and/or physical means, so long as thetechnique does not deleteriously affect the properties of the toxin, nordiminish the cellular capability in protecting the toxin. Examples ofchemical reagents are halogenating agents, particularly halogens ofatomic no. 17-80. More particularly, iodine can be used under mildconditions and for sufficient time to achieve the desired results. Othersuitable techniques include treatment with aldehydes, such asformaldehyde and glutaraldehye; anti-infectives, such as zephiranchloride and cetylpyridinium chloride; alcohols, such as isopropyl andethanol; various histologic fixatives, such as Lugol's iodine, Bouin'sfixative, and Helly's fixatives, (see e.g., Humason, 1967); or acombination of physical (heat) and chemical agents that preserve andprolong the activity of the toxin produced in the cell when the cell isadministered to the host animal. Examples of physical means are shortwavelength radiation such as γ-radiation and X-radiation, freezing, UVirradiation, lyophilization, and the like. The cells employed willusually be intact and be substantially in the proliferative form whentreated, rather than in a spore form, although in some instances sporesmay be employed.

[0183] Where the B. thuringiensis toxin gene is introduced via asuitable vector into a microbial host, and said host is applied to theenvironment in a living state, it is essential that certain hostmicrobes be used. Microorganism hosts are selected which are known tooccupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment (crop and other insect habitats) with the wild-typemicroorganisms, provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, provide forimproved protection of the pesticide from environmental degradation andinactivation.

[0184] A large number of microorganisms are known to inhabit thephylloplane (the surface of the plant leaves) and/or the rhizosphere(the soil surrounding plant roots) of a wide variety of important crops.These microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Bacillus(including the species and subspecies B. thuringiensis kurstaki HD-1, B.thuringiensis kurstaki HD-73, B. thuringiensis sotto, B. thuringiensisberliner, B. thuringiensis thuringiensis, B. thuringiensis tolworthi, B.thuringiensis dendrolimus, B. thuringiensis alesti, B. thuringiensisgalleriae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B.thuringiensis entomocidus, B. thuringiensis tenebrionis and B.thuringiensis san diego); Pseudomonas, Erwinia, Serratia, Klebsiella,Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., generaSaccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula,and Aureobasidium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobactersphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligeneseutrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans.

[0185] 4.4 Definitions

[0186] In accordance with the present invention, nucleic acid sequencesinclude and are not limited to DNA (including and not limited to genomicor extragenomic DNA), genes, RNA (including and not limited to mRNA andtRNA), nucleosides, and suitable nucleic acid segments either obtainedfrom native sources, chemically synthesized, modified, or otherwiseprepared by the hand of man. The following words and phrases have themeanings set forth below.

[0187] A, an: In accordance with long standing patent law convention,the words “a” and “an” when used in this application, including theclaims, denotes “one or more”.

[0188] Broad-spectrum: Refers to a wide range of insect species.

[0189] Broad-spectrum activity: The toxicity towards a wide range ofinsect species.

[0190] Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

[0191] Insecticidal activity: The toxicity towards insects.

[0192] Insecticidal specificity: The toxicity exhibited by a crystalprotein or proteins, microbe or plant, towards multiple insect species.

[0193] Intraorder specificity: The toxicity of a particular crystalprotein towards insect species within an Order of insects (e.g., OrderColeoptera).

[0194] Interorder specificity: The toxicity of a particular crystalprotein towards insect species of different Orders (e.g., OrdersColeoptera and Diptera).

[0195] LC₅₀: The lethal concentration of crystal protein that causes 50%mortality of the insects treated.

[0196] LC₉₅: The lethal concentration of crystal protein that causes 95%mortality of the insects treated.

[0197] Promoter: A recognition site on a DNA sequence or group of DNAsequences that provide an expression control element for a structuralgene and to which RNA polymerase specifically binds and initiates RNAsynthesis (transcription) of that gene.

[0198] Regeneration: The process of growing a plant from a plant cell(e.g., plant protoplast or explant).

[0199] Structural gene: A gene that is expressed to produce apolypeptide.

[0200] Transformation: A process of introducing an exogenous DNAsequence (e.g., a vector, a recombinant DNA molecule) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

[0201] Transformed cell: A cell whose DNA has been altered by theintroduction of an exogenous DNA molecule into that cell.

[0202] Transgenic cell: Any cell derived or regenerated from atransformed cell or derived from a transgenic cell. Exemplary transgeniccells include plant calli derived from a transformed plant cell andparticular cells such as leaf, root, stem, e.g., somatic cells, orreproductive (germ) cells obtained from a transgenic plant.

[0203] Transgenic plant: A plant or progeny thereof derived from atransformed plant cell or protoplast, wherein the plant DNA contains anintroduced exogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant whose DNA contains an exogenous DNAmolecule. However, it is thought more scientifically correct to refer toa regenerated plant or callus obtained from a transformed plant cell orprotoplast as being a transgenic plant, and that usage will be followedherein.

[0204] Vector: A DNA molecule capable of replication in a host celland/or to which another DNA segment can be operatively linked so as tobring about replication of the attached segment. A plasmid is anexemplary vector.

[0205] As used herein, the designations “CryIII” and “Cry3” aresynonymous, as are the designations “CryIIIB2” and “Cry3Bb.” Likewise,the inventors have utilized the generic term Cry3Bb* to denote any andall Cry3Bb variants which comprise amino acid sequences modified in theprotein. Similarly, cry3Bb* is meant to denote any and all nucleic acidsegments and/or genes which encode a Cry3Bb* protein, etc.

[0206] 4.5 Preparation of Cry3* Polynucleotides

[0207] Once the structure of the desired peptide to be mutagenized hasbeen analyzed using one or more of the design strategies disclosedherein, it will be desirable to introduce one or more mutations intoeither the protein or, alternatively, into the DNA sequence encoding theprotein for the purpose of producing a mutated protein with alteredbioinsecticidal properties.

[0208] To that end, the present invention encompasses both site-specificmutagenesis methods and random mutagenesis of a nucleic acid segmentencoding a crystal protein in the manner described herein. Inparticular, methods are disclosed for the mutagenesis of nucleic acidsegments encoding the amino acid sequences using one or more of thedesign strategies described herein. Using the assay methods describedherein, one may then identify mutants arising from these procedureswhich have improved insecticidal properties or altered specificity,either intraorder or interorder.

[0209] The means for mutagenizing a DNA segment encoding a crystalprotein are well-known to those of skill in the art. Modifications maybe made by random, or site-specific mutagenesis procedures. The nucleicacid may be modified by altering its structure through the addition ordeletion of one or more nucleotides from the sequence.

[0210] Mutagenesis may be performed in accordance with any of thetechniques known in the art such as and not limited to synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular crystal protein. A “suitable host” is any host which willexpress Cry3Bb, such as and not limited to B. thuringiensis and E. coli.Screening for insecticidal activity, in the case of Cry3Bb includes andis not limited to coleopteran-toxic activity which may be screened forby techniques known in the art.

[0211] In particular, site-specific mutagenesis is a technique useful inthe preparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, for example, incorporating one ormore of the foregoing considerations, by introducing one or morenucleotide sequence changes into the DNA. Site-specific mutagenesisallows the production of mutants through the use of specificoligonucleotide sequences which encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 17 to about 75 nucleotides ormore in length is preferred, with about 10 to about 25 or more residueson both sides of the junction of the sequence being altered.

[0212] In general, the technique of site-specific mutagenesis is wellknown in the art, as exemplified by various publications. As will beappreciated, the technique typically employs a phage vector which existsin both a single stranded and double stranded form. Typical vectorsuseful in site-directed mutagenesis include vectors such as the M13phage. These phage are readily commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis whicheliminates the step of transferring the gene of interest from a plasmidto a phage.

[0213] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform or transfect appropriate cells, such as E. colicells, and clones are selected which include recombinant vectors bearingthe mutated sequence arrangement. A genetic selection scheme was devisedby Kunkel et al. (1987) to enrich for clones incorporating the mutagenicoligonucleotide. Alternatively, the use of PCR™ with commerciallyavailable thermostable enzymes such as Taq polymerase may be used toincorporate a mutagenic oligonucleotide primer into an amplified DNAfragment that can then be cloned into an appropriate cloning orexpression vector. The PCR™-mediated mutagenesis procedures of Tomic etal. (1990) and Upender et al. (1995) provide two examples of suchprotocols. A PCR™ employing a thermostable ligase in addition to athermostable polymerase may also be used to incorporate a phosphorylatedmutagenic oligonucleotide into an amplified DNA fragment that may thenbe cloned into an appropriate cloning or expression vector. Themutagenesis procedure described by Michael (1994) provides an example ofone such protocol.

[0214] The preparation of sequence variants of the selectedpeptide-encoding DNA segments using site-directed mutagenesis isprovided as a means of producing potentially useful species and is notmeant to be limiting as there are other ways in which sequence variantsof peptides and the DNA sequences encoding them may be obtained. Forexample, recombinant vectors encoding the desired peptide sequence maybe treated with mutagenic agents, such as hydroxylamine, to obtainsequence variants.

[0215] As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety

[0216] A number of template dependent processes are available to amplifythe target sequences of interest present in a sample. One of the bestknown amplification methods is the polymerase chain reaction (PCR™)which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159 (each of which is specifically incorporated herein byreference in its entirety). Briefly, in PCR™, two primer sequences areprepared which are complementary to regions on opposite complementarystrands of the target sequence. An excess of deoxynucleosidetriphosphates are added to a reaction mixture along with a DNApolymerase (e.g., Taq polymerase). If the target sequence is present ina sample, the primers will bind to the target and the polymerase willcause the primers to be extended along the target sequence by adding onnucleotides. By raising and lowering the temperature of the reactionmixture, the extended primers will dissociate from the target to formreaction products, excess primers will bind to the target and to thereaction products and the process is repeated. Preferably a reversetranscriptase PCR™ amplification procedure may be performed in order toquantify the amount of mRNA amplified. Polymerase chain reactionmethodologies are well known in the art.

[0217] Another method for amplification is the ligase chain reaction(referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308,incorporated herein by reference in its entirety. In LCR, twocomplementary probe pairs are prepared, and in the presence of thetarget sequence, each pair will bind to opposite complementary strandsof the target such that they abut. In the presence of a ligase, the twoprobe pairs will link to form a single unit. By temperature cycling, asin PCR™, bound ligated units dissociate from the target and then serveas “target sequences” for ligation of excess probe pairs. U.S. Pat. No.4,883,750, specifically incorporated herein by reference in itsentirety, describes an alternative method of amplification similar toLCR for binding probe pairs to a target sequence.

[0218] Qbeta Replicase™, described in Intl. Pat. Appl. Publ. No.PCT/US87/00880, incorporated herein by reference in its entirety, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA which has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence which can then be detected.

[0219] An isothermal amplification method, in which restrictionendonucleases and ligases are used to achieve the amplification oftarget molecules that contain nucleotide 5′-[α-thio]triphosphates in onestrand of a restriction site (Walker et al., 1992, incorporated hereinby reference in its entirety), may also be useful in the amplificationof nucleic acids in the present invention.

[0220] Strand Displacement Amplification (SDA) is another method ofcarrying out isothermal amplification of nucleic acids which involvesmultiple rounds of strand displacement and synthesis, i.e., nicktranslation. A similar method, called Repair Chain Reaction (RCR) isanother method of amplification which may be useful in the presentinvention and is involves annealing several probes throughout a regiontargeted for amplification, followed by a repair reaction in which onlytwo of the four bases are present. The other two bases can be added asbiotinylated derivatives for easy detection. A similar approach is usedin SDA

[0221] Sequences can also be detected using a cyclic probe reaction(CPR). In CPR, a probe having 3′ and 5′ end sequences ofnon-Cry-specific DNA and an internal sequence of a Cry-specific RNA ishybridized to DNA which is present in a sample. Upon hybridization, thereaction is treated with RNaseH, and the products of the probeidentified as distinctive products generating a signal which arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated. Thus, CPR involvesamplifying a signal generated by hybridization of a probe to acry-specific expressed nucleic acid

[0222] Still other amplification methods described in Great Britain Pat.Appl. No. 2 202 328, and in Intl. Pat. Appl. Publ. No. PCT/US89/01025,each of which is incorporated herein by reference in its entirety, maybe used in accordance with the present invention. In the formerapplication, “modified” primers are used in a PCR™ like, template andenzyme dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence

[0223] Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (Kwoh et al., 1989;Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by referencein its entirety), including nucleic acid sequence based amplification(NASBA) and 3SR. In NASBA, the nucleic acids can be prepared foramplification by standard phenol/chloroform extraction, heatdenaturation of a sample, treatment with lysis buffer and minispincolumns for isolation of DNA and RNA or guanidinium chloride extractionof RNA. These amplification techniques involve annealing a primer whichhas crystal protein-specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second crystalprotein-specific primer, followed by polymerization. The double strandedDNA molecules are then multiply transcribed by a polymerase such as T7or SP6. In an isothermal cyclic reaction, the RNAs are reversetranscribed into double stranded DNA, and transcribed once against witha polymerase such as T7 or SP6. The resulting products, whethertruncated or complete, indicate crystal protein-specific sequences.

[0224] Eur. Pat. Appl. Publ. No. 329,822, incorporated herein byreference in its entirety, disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H (RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA

[0225] Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein byreference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic; i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” (Frohman, 1990),and “one-sided PCR™” (Ohara, 1989) which are well-known to those ofskill in the art.

[0226] Methods based on ligation of two (or more) oligonucleotides inthe presence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide (Wu andDean, 1996, incorporated herein by reference in its entirety), may alsobe used in the amplification of DNA sequences of the present invention.

[0227] 4.6 Phage-Resistant Variants

[0228] In certain embodiments, one may desired to prepare one or morephage resistant variants of the B. thuringiensis mutants prepared by themethods described herein. To do so, an aliquot of a phage lysate isspread onto nutrient agar and allowed to dry. An aliquot of the phagesensitive bacterial strain is then plated directly over the dried lysateand allowed to dry. The plates are incubated at 30° C. The plates areincubated for 2 days and, at that time, numerous colonies could be seengrowing on the agar. Some of these colonies are picked and subculturedonto nutrient agar plates. These apparent resistant cultures are testedfor resistance by cross streaking with the phage lysate. A line of thephage lysate is streaked on the plate and allowed to dry. Thepresumptive resistant cultures are then streaked across the phage line.Resistant bacterial cultures show no lysis anywhere in the streak acrossthe phage line after overnight incubation at 30° C. The resistance tophage is then reconfirmed by plating a lawn of the resistant cultureonto a nutrient agar plate. The sensitive strain is also plated in thesame manner to serve as the positive control. After drying, a drop ofthe phage lysate is plated in the center of the plate and allowed todry. Resistant cultures showed no lysis in the area where the phagelysate has been placed after incubation at 30° C. for 24 hours.

[0229] 4.7 Crystal Protein Compositions as Insecticides and Methods ofUse

[0230] Order Coleoptera comprises numerous beetle species includingground beetles, reticulated beetles, skin and larder beetles,long-horned beetles, leaf beetles, weevils, bark beetles, ladybirdbeetles, soldier beetles, stag beetles, water scavenger beetles, and ahost of other beetles. A brief taxonomy of the Order is given at thewebsite http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html.

[0231] Particularly important among the Coleoptera are the agriculturalpests included within the infraorders Chrysomeliformia and Cucujiformia.Members of the infraorder Chrysomeliformia, including the leaf beetles(Chrysomelidae) and the weevils (Curculionidae), are particularlyproblematic to agriculture, and are responsible for a variety of insectdamage to crops and plants. The infraorder Cucujiformia includes thefamilies Coccinellidae, Cucujidae, Lagridae, Meloidae, Rhipiphoridae,and Tenebrionidae. Within this infraorder, members of the familyChrysomelidae (which includes the genera Exema, Chrysomela, Oreina,Chrysolina, Leptinotarsa, Gonioctena, Oulema, Monozia, Ophraella,Cerotoma, Diabrotica, and Lachnaia), are well-known for their potentialto destroy agricultural crops.

[0232] As the toxins of the present invention have been shown to beeffective in combatting a variety of members of the order Coleoptera,the inventors contemplate that the insects of many Coleopteran generamay be controlled or eradicated using the polypeptide compositionsdescribed herein. Likewise, the methods described herein for generatingmodified polypeptides having enhanced insect specificity may also beuseful in extending the range of the insecticidal activity of themodified polypeptides to other insect species within, and outside of,the Order Coleoptera.

[0233] As such, the inventors contemplate that the crystal proteincompositions disclosed herein will find particular utility asinsecticides for topical and/or systemic application to field crops,including but not limited to rice, wheat, alfalfa, corn (maize),soybeans, tobacco, potato, barley, canola (rapeseed), sugarbeet,sugarcane, flax, rye, oats, cotton, sunflower; grasses, such as pastureand turf grasses; fruits, citrus, nuts, trees, shrubs and vegetables; aswell as ornamental plants, cacti, succulents, and the like.

[0234] Disclosed and claimed is a composition comprising aninsecticidally-effective amount of a Cry3Bb* crystal proteincomposition. The composition preferably comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:100, or SEQ ID NO:108 or biologically-functionalequivalents thereof.

[0235] The insecticide composition may also comprise a Cry3Bb* crystalprotein that is encoded by a nucleic acid sequence having the sequenceof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNQ:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO;65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:99, or SEQ ID NO:108, or, alternatively, a nucleic acid sequencewhich hybridizes to the nucleic acid sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13.SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, or SEQ ID NO:107 under conditions of moderate stringency.

[0236] The insecticidal compositions may comprise one or more B.thuringiensis cell types, or one or more cultures of such cells, or,alternatively, a mixture of one or more B. thuringiensis cells whichexpress one or more of the novel crystal proteins of the invention incombination with another insecticidal composition. In certain aspects itmay be desirable to prepare compositions which contain a plurality ofcrystal proteins, either native or modified, for treatment of one ormore types of susceptible insects. The B. thuringiensis cells of theinvention can be treated prior to formulation to prolong theinsecticidal activity when the cells are applied to the environment ofthe target insect(s). Such treatment can be by chemical or physicalmeans, or by a combination of chemical and/or physical means, so long asthe technique does not deleteriously affect the properties of theinsecticide, nor diminish the cellular capability in protecting theinsecticide. Examples of chemical reagents are halogenerating agents,particularly halogens of atomic no. 17-80. More particularly, iodine canbe used under mild conditions and for sufficient time to achieve thedesired results. Other suitable techniques include treatment withaldehydes, such as formaldehyde and glutaraldehyde; anti-infectives,such as zephiran chloride; alcohols, such as isopropyl and ethanol;various histologic fixatives, such as Bouin's fixative and Helly'sfixative (see Humason, 1967); or a combination of physical (heat) andchemical agents that prolong the activity of the δ-endotoxin produced inthe cell when the cell is applied to the environment of the targetpest(s). Examples of physical means are short wavelength radiation suchas gamma-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like.

[0237] The inventors contemplate that any formulation methods known tothose of skill in the art may be employed using the proteins disclosedherein to prepare such bioinsecticide compositions. It may be desirableto formulate whole cell preparations, cell extracts, cell suspensions,cell homogenates, cell lysates, cell supernatants, cell filtrates, orcell pellets of a cell culture (preferably a bacterial cell culture suchas a B. thuringiensis cell culture described in Table 3) that expressesone or more cry3Bb* DNA segments to produce the encoded Cry3Bb*protein(s) or peptide(s). The methods for preparing such formulationsare known to those of skill in the art, and may include, e.g.,desiccation, lyophilization, homogenization, extraction, filtration,centrifugation, sedimentation, or concentration of one or more culturesof bacterial cells, such as B. thuringiensis cells described in Table 3,which express the Cry3Bb* peptide(s) of interest.

[0238] In one preferred embodiment, the bioinsecticide compositioncomprises an oil flowable suspension comprising lysed or unlysedbacterial cells, spores, or crystals which contain one or more of thenovel crystal proteins disclosed herein. Preferably the cells are B.thuringiensis cells, however, any such bacterial host cell expressingthe novel nucleic acid segments disclosed herein and producing a crystalprotein is contemplated to be useful, such as Bacillus spp., includingB. megaterium, B. subtilis; B. cereus, Escherichia spp., including E.coli, and/or Pseudomonas spp., including P. cepacia, P. aeruginosa, andP. fluorescens. Alternatively, the oil flowable suspension may consistof a combination of one or more of the following compositions: lysed orunlysed bacterial cells, spores, crystals, and/or purified crystalproteins.

[0239] In a second preferred embodiment, the bioinsecticide compositioncomprises a water dispersible granule or powder. This granule or powdermay comprise lysed or unlysed bacterial cells, spores, or crystals whichcontain one or more of the novel crystal proteins disclosed herein.Preferred sources for these compositions include bacterial cells such asB. thuringiensis cells, however, bacteria of the genera Bacillus,Escherichia, and Pseudomonas which have been transformed with a DNAsegment disclosed herein and expressing the crystal protein are alsocontemplated to be useful. Alternatively, the granule or powder mayconsist of a combination of one or more of the following compositions:lysed or unlysed bacterial cells, spores, crystals, and/or purifiedcrystal proteins.

[0240] In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, spray, emulsion, colloid, aqueous ororganic solution, dust, pellet, or collodial concentrate. Such acomposition may contain either unlysed or lysed bacterial cells, spores,crystals, or cell extracts as described above, which contain one or moreof the novel crystal proteins disclosed herein. Preferred bacterialcells are B. thuringiensis cells, however, bacteria such as B.megaterium, B. subtilis, B. cereus, E. coli, or Pseudomonas spp. cellstransformed with a DNA segment disclosed herein and expressing thecrystal protein are also contemplated to be useful. Such dry forms ofthe insecticidal compositions may be formulated to dissolve immediatelyupon wetting, or alternatively, dissolve in a controlled-release,sustained-release, or other time-dependent manner. Alternatively, such acomposition may consist of a combination of one or more of the followingcompositions: lysed or unlysed bacterial cells, spores, crystals, and/orpurified crystal proteins.

[0241] In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous solution or suspension or cell culture of lysed orunlysed bacterial cells, spores, crystals, or a mixture of lysed orunlysed bacterial cells, spores, and/or crystals, such as thosedescribed above which contain one or more of the novel crystal proteinsdisclosed herein. Such aqueous solutions or suspensions may be providedas a concentrated stock solution which is diluted prior to application,or alternatively, as a diluted solution ready-to-apply.

[0242] For these methods involving application of bacterial cells, thecellular host containing the Crystal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

[0243] When the insecticidal compositions comprise B. thuringiensiscells, spores, and/or crystals containing the modified crystalprotein(s) of interest, such compositions may be formulated in a varietyof ways. They may be employed as wettable powders, granules or dusts, bymixing with various inert materials, such as inorganic minerals(phyllosilicates, carbonates, sulfates, phosphates, and the like) orbotanical materials (powdered corncobs, rice hulls, walnut shells, andthe like). The formulations may include spreader-sticker adjuvants,stabilizing agents, other pesticidal additives, or surfactants. Liquidformulations may be aqueous-based or non-aqueous and employed as foams,suspensions, emulsifiable concentrates, or the like. The ingredients mayinclude rheological agents, surfactants, emulsifiers, dispersants, orpolymers.

[0244] Alternatively, the novel Cry3Bb-derived mutated crystal proteinsmay be prepared by native or recombinant bacterial expression systems invitro and isolated for subsequent field application. Such protein may beeither in crude cell lysates, suspensions, colloids, etc., oralternatively may be purified, refined, buffered, and/or furtherprocessed, before formulating in an active biocidal formulation.Likewise, under certain circumstances, it may be desirable to isolatecrystals and/or spores from bacterial cultures expressing the crystalprotein and apply solutions, suspensions, or collodial preparations ofsuch crystals and/or spores as the active bioinsecticidal composition.

[0245] Another important aspect of the invention is a method ofcontrolling coleopteran insects which are susceptible to the novelcompositions disclosed herein. Such a method generally comprisescontacting the insect or insect population, colony, etc., with aninsecticidally-effective amount of a Cry3Bb* crystal proteincomposition. The method may utilize Cry3Bb* crystal proteins such asthose disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,SEQ ID NO:70, SEQ ID NO:100, or SEQ ID NO:108, or biologicallyfunctional equivalents thereof.

[0246] Alternatively, the method may utilize one or more Cry3Bb* crystalproteins which are encoded by the nucleic acid sequences of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ IDNO:101, or SEQ ID NO:107, or by one or more nucleic acid sequences whichhybridize to the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107,under conditions of moderate, or higher, stringency. The methods foridentifying sequences which hybridize to those disclosed underconditions of moderate or higher stringency are well-known to those ofskill in the art, and are discussed herein.

[0247] Regardless of the method of application, the amount of the activecomponent(s) are applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

[0248] The insecticide compositions described may be made by formulatingeither the bacterial cell, crystal and/or spore suspension, or isolatedprotein component with the desired agriculturally-acceptable carrier.The compositions may be formulated prior to administration in anappropriate means such as lyophilized, freeze-dried, dessicated, or inan aqueous carrier, medium or suitable diluent, such as saline or otherbuffer. The formulated compositions may be in the form of a dust orgranular material, or a suspension in oil (vegetable or mineral), orwater or oil/water emulsions, or as a wettable powder, or in combinationwith any other carrier material suitable for agricultural application.Suitable agricultural carriers can be solid or liquid and are well knownin the art. The term “agriculturally-acceptable carrier” covers alladjuvants, e.g., inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in insecticide formulationtechnology; these are well known to those skilled in insecticideformulation. The formulations may be mixed with one or more solid orliquid adjuvants and prepared by various means, e.g., by homogeneouslymixing, blending and/or grinding the insecticidal composition withsuitable adjuvants using conventional formulation techniques.

[0249] The insecticidal compositions of this invention are applied tothe environment of the target coleopteran insect, typically onto thefoliage of the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

[0250] Other application techniques, e.g., dusting, sprinkling, soaking,soil injection, soil tilling, seed coating, seedling coating, spraying,aerating, misting, atomizing, and the like, are also feasible and may berequired under certain circumstances such as e.g., insects that causeroot or stalk infestation, or for application to delicate vegetation orornamental plants. These application procedures are also well-known tothose of skill in the art.

[0251] The insecticidal composition of the invention may be employed inthe method of the invention singly or in combination with othercompounds, including and not limited to other pesticides. The method ofthe invention may also be used in conjunction with other treatments suchas surfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

[0252] The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 1% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 1% to about 99% or more by weight ofthe composition, while liquid formulations may generally comprise fromabout 1% to about 99% or more of the active ingredient by weight.Formulations which comprise intact bacterial cells will generallycontain from about 10⁴ to about 10¹² cells/mg

[0253] The insecticidal formulation may be administered to a particularplant or target area in one or more applications as needed, with atypical field application rate per hectare ranging on the order of fromabout 1 g to about 1 kg, 2 kg, 5, kg, or more of active ingredient.

[0254] 4.8 Nucleic Acid Segments as Hybridization Probes and Primers

[0255] In addition to their use in directing the expression of crystalproteins or peptides of the present invention, the nucleic acidsequences contemplated herein also have a variety of other uses. Forexample, they also have utility as probes or primers in nucleic acidhybridization embodiments. As such, it is contemplated that nucleic acidsegments that comprise a sequence region that consists of at least a 14nucleotide long contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99,SEQ ID NO:101, or SEQ ID NO:107 will find particular utility. Longercontiguous identical or complementary sequences, e.g., those of about20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, 10000 etc. (includingall intermediate lengths and up to and including full-length sequenceswill also be of use in certain embodiments.

[0256] The ability of such nucleic acid probes to specifically hybridizeto crystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

[0257] Nucleic acid molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so, identical or complementary to DNA sequencesof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:99, SEQ ID NO:101, or SEQ ID NO:107 are particularly contemplated ashybridization probes for use in, e.g., Southern and Northern blotting.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 10-14 and about 100 or 200 nucleotides, but largercontiguous complementary stretches may be used, according to the lengthcomplementary sequences one wishes to detect.

[0258] The use of a hybridization probe of about 14 nucleotides inlength allows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 14 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 20 contiguous nucleotides,or even longer where desired.

[0259] Of course, fragments may also be obtained by other techniquessuch as, e.g., by mechanical shearing or by restriction enzymedigestion. Small nucleic acid segments or fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, as is commonly practiced using an automated oligonucleotidesynthesizer. Also, fragments may be obtained by application of nucleicacid reproduction technology, such as the PCR™ technology of U.S. Pat.Nos. 4,683,195 and 4,683,202 (each incorporated herein by reference), byintroducing selected sequences into recombinant vectors for recombinantproduction, and by other recombinant DNA techniques generally known tothose of skill in the art of molecular biology.

[0260] Accordingly, the nucleotide sequences of the invention may beused for their ability to selectively form duplex molecules withcomplementary stretches of DNA fragments. Depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C. Such selectiveconditions tolerate little, if any, mismatch between the probe and thetemplate or target strand, and would be particularly suitable forisolating crystal protein-encoding DNA segments. Detection of DNAsegments via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (eachincorporated herein by reference) are exemplary of the methods ofhybridization analyses. Teachings such as those found in the texts ofMaloy et al., 1994; Segal 1976; Prokop, 1991; and Kuby, 1994, areparticularly relevant.

[0261] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0262] In certain embodiments, it will be advantageous to employ nucleicacid sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of giving a detectable signal. Inpreferred embodiments, one will likely desire to employ a fluorescentlabel or an enzyme tag, such as urease, alkaline phosphatase orperoxidase, instead of radioactive or other environmental undesirablereagents. In the case of enzyme tags, colorimetric indicator substratesare known that can be employed to provide a means visible to the humaneye or spectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

[0263] In general, it is envisioned that the hybridization probesdescribed herein will be useful both as reagents in solutionhybridization as well as in embodiments employing a solid phase. Inembodiments involving a solid phase, the test DNA (or RNA) is adsorbedor otherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Following washing of the hybridized surface so as toremove nonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

[0264] 4.9 Characters of Modified Cry3 δ-Endotoxins

[0265] The present invention provides novel polypeptides that define awhole or a portion of a B. thuringiensis cry3Bb.60, cry3Bb.11221,cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225, cry3Bb.11226,cry3Bb.11227, cry3Bb.11228, cry3Bb.11229, cry3Bb.11230, cry3Bb.11231,cry3Bb.11232, cry3Bb.11233, cry3Bb.11234, cry3Bb.11235, cry3Bb.11236,cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241, cry3Bb.11242,cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046, cry3Bb.11048,cry3Bb.11051, cry3Bb.11057, cry3Bb.11058, cry3Bb.11081, cry3Bb.11082,cry3Bb.11083, cry3Bb.11084, cry3Bb.11095 and cry3Bb.11098-encodedcrystal protein.

[0266] 4.10 Crystal Protein Nomenclature

[0267] The inventors have arbitrarily assigned the designationsCry3Bb.60, Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224,Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228, Cry3Bb.11229,Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234,Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239,Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036,Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057, Cry3Bb.11058,Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11095 andCry3Bb.11098 to the novel proteins of the invention.

[0268] Likewise, the arbitrary designations of cry3Bb.60, cry3Bb.11221,cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225, cry3Bb.11226,cry3Bb.11227, cry3Bb.11228, cry3Bb.11229, cry3Bb.11230, cry3Bb.11231,cry3Bb.11232, cry3Bb.11233, cry3Bb.11234, cry3Bb.11235, cry3Bb.11236,cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241, cry3Bb.11242,cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046, cry3Bb.11048,cry3Bb.11051, cry3Bb.11057, cry3Bb.11058, cry3Bb.11081, cry3Bb.11082,cry3Bb.11083, cry3Bb.11084, cry3Bb.11095 and Cry3Bb.11098 have beenassigned to the novel nucleic acid sequences which encode thesepolypeptides, respectively. While formal assignment of gene and proteindesignations based on the revised nomenclature of crystal proteinendotoxins (Table 1) may be made by the committee on the nomenclature ofB. thuringiensis. any re-designations of the compositions of the presentinvention are also contemplated to be fully within the scope of thepresent disclosure.

[0269] 4.11 Transformed Host Cells and Transgenic Plants

[0270] A bacterium, a yeast cell, or a plant cell or a plant transformedwith an expression vector of the present invention is also contemplated.A transgenic bacterium, yeast cell, plant cell or plant derived fromsuch a transformed or transgenic cell is also one aspect of theinvention.

[0271] Such transformed host cells are often desirable for use in theproduction of endotoxins and for expression of the various DNA geneconstrcuts disclosed herein. In some aspects of the invention, it isoften desirable to modulate, regulate, or otherwise control theexpression of the gene segments disclosed herein. Such methods areroutine to those of skill in the molecular genetic arts. Typically, whenincreased or over-expression of a particular gene is desired, variousmanipulations may be employed for enhancing the expression of themessenger RNA, particularly by using an active promoter, as well as byemploying sequences, which enhance the stability of the messenger RNA inthe particular transformed host cell.

[0272] Typically, the initiation and translational termination regionwill involve stop codon(s), a terminator region, and optionally, apolyadenylation signal. In the direction of transcription, namely in the5′ to 3′ direction of the coding or sense sequence, the construct willinvolve the transcriptional regulatory region, if any, and the promoter,where the regulatory region may be either 5′ or 3′ of the promoter, theribosomal binding site, the initiation codon, the structural gene havingan open reading frame in phase with the initiation codon, the stopcodon(s), the polyadenylation signal sequence, if any, and theterminator region. This sequence as a double strand may be used byitself for transformation of a microorganism host, but will usually beincluded with a DNA sequence involving a marker, where the second DNAsequence may be joined to the δ-endotoxin expression construct duringintroduction of the DNA into the host.

[0273] By a marker is intended a structural gene which provides forselection of those hosts which have been modified or transformed. Themarker will normally provide for selective advantage, for example,providing for biocide resistance, e.g., resistance to antibiotics orheavy metals; complementation, so as to provide prototropy to anauxotrophic host, or the like. Preferably, complementation is employed,so that the modified host may not only be selected, but may also becompetitive in the field. One or more markers may be employed in thedevelopment of the constructs, as well as for modifying the host. Theorganisms may be further modified by providing for a competitiveadvantage against other wild-type microorganisms in the field. Forexample, genes expressing metal chelating agents, e.g., siderophores,may be introduced into the host along with the structural geneexpressing the δ-endotoxin. In this manner, the enhanced expression of asiderophore may provide for a competitive advantage for theδ-endotoxin-producing host, so that it may effectively compete with thewild-type microorganisms and stably occupy a niche in the environment.

[0274] Where no functional replication system is present, the constructwill also include a sequence of at least 50 basepairs (bp), preferablyat least about 100 bp, and usually not more than about 1000 bp of asequence homologous with a sequence in the host. In this way, theprobability of legitimate recombination is enhanced, so that the genewill be integrated into the host and stably maintained by the host.Desirably, the δ-endotoxin gene will be in close proximity to the geneproviding for complementation as well as the gene providing for thecompetitive advantage. Therefore, in the event that a δ-endotoxin geneis lost, the resulting organism will be likely to also lose thecomplementing gene and/or the gene providing for the competitiveadvantage, so that it will be unable to compete in the environment withthe gene retaining the intact construct.

[0275] The crystal protein-encoding gene can be introduced between thetranscriptional and translational initiation region and thetranscriptional and translational termination region, so as to be underthe regulatory control of the initiation region. This construct will beincluded in a plasmid, which will include at least one replicationsystem, but may include more than one, where one replication system isemployed for cloning during the development of the plasmid and thesecond replication system is necessary for functioning in the ultimatehost. In addition, one or more markers may be present, which have beendescribed previously. Where integration is desired, the plasmid willdesirably include a sequence homologous with the host genome.

[0276] The transformants can be isolated in accordance with conventionalways, usually employing a selection technique, which allows forselection of the desired organism as against unmodified organisms ortransferring organisms when present. The transformants then can betested for pesticidal activity.

[0277] Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the δ-endotoxin in the cell when thethen treated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the δ-endotoxin is unstable or thelevel of application sufficiently low as to avoid any possibility oftoxicity to a mammalian host. As hosts, of particular interest will bethe prokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobitim; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibdo, Spirillum; Lactobacillaceae; phylloplane organisms such asmembers of the Pseudomonadaceae (including Pseudomonas spp. andAcetobacter spp.); Azotobacteraceae and Nitrobacteraceae; Flavobacteriumspp.; members of the Bacillaceae such as Lactobacillus spp.,Bifidobacterium, and Bacillus spp., and the like. Particularly preferredhost cells include Pseudomonas aeruginosa, Pseudomonas fluorescens,Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and thelike.

[0278] Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes,which includes yeast, such as Schizosaccharomyces; and Basidiomycetes,Rhodotorula, Aureobasidium, Sporobolomyces, Saccharomyces spp., andSporobolomyces spp.

[0279] Characteristics of particular interest in selecting a host cellfor purposes of production include ease of introducing the δ-endotoxingene into the host, availability of expression systems, efficiency ofexpression, stability of the pesticide in the host, and the presence ofauxiliary genetic capabilities. Characteristics of interest for use as apesticide micro-capsule include protective qualities for the pesticide,such as thick cell walls, pigmentation, and intracellular packaging orformation of inclusion bodies; leaf affinity; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the δ-endotoxin; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

[0280] The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed. Treatment of the recombinantmicrobial cell can be done as disclosed infra. The treated cellsgenerally will have enhanced structural stability which will enhanceresistance to environmental conditions.

[0281] Genes or other nucleic acid segments, as disclosed herein, can beinserted into host cells using a variety of techniques which are wellknown in the art. For example, a large number of cloning vectorscomprising a replication system in E. coli and a marker that permitsselection of the transformed cells are available for preparation for theinsertion of foreign genes into higher organisms, including plants. Thevectors comprise, for example, pBR322, pUC series, M13mp series,pACYC184, etc. Accordingly, the sequence coding for the δ-endotoxin canbe inserted into the vector at a suitable restriction site. Theresulting plasmid is used for transformation into E. coli. The E. colicells are cultivated in a suitable nutrient medium, then harvested andlysed. The plasmid is recovered. Sequence analysis, restrictionanalysis, electrophoresis, and other biochemical-molecular biologicalmethods are generally carried out as methods of analysis. After eachmanipulation, the DNA sequence used can be cleaved and joined to thenext DNA sequence. Each plasmid sequence can be cloned in the same orother plasmids. Depending on the method of inserting desired genes intothe plant, other DNA sequences may be necessary.

[0282] Methods for DNA transformation of plant cells includeAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos and particle bombardment. Each of these methodshas distinct advantages and disadvantages. Thus, one particular methodof introducing genes into a particular plant strain may not necessarilybe the most effective for another plant strain, but it is well knownwhich methods are useful for a particular plant strain.

[0283] Suitable methods are believed to include virtually any method bywhich DNA can be introduced into a cell, such as by Agrobacteriuminfection, direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

[0284] Technology for introduction of DNA into cells is well-known tothose of skill in the art. Four general methods for delivering a geneinto cells have been described: (1) chemical methods (Graham and van derEb, 1973; Zatloukal et al., 1992); (2) physical methods such asmicroinjection (Capecchi, 1980), electroporation (Wong and Neumann,1982; Fromm et al., 1985) and the gene gun (Johnston and Tang, 1994;Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993;Eglitis and Anderson, 1988; Eglitis et al., 1988); and (4)receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner et al.,1992).

[0285] A large number of techniques are available for inserting DNA intoa plant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobactedum rhizogenes astransformation agent, fusion, injection, or electroporation as well asother possible methods. If agrobacteria are used for the transformation,the DNA to be inserted has to be cloned into special plasmids, namelyeither into an intermediate vector or into a binary vector. Theintermediate vectors can be integrated into the Ti or Ri plasmid byhomologous recombination owing to sequences that are homologous tosequences in the T-DNA. The Ti or Ri plasmid also comprises the virregion necessary for the transfer of the T-DNA.

[0286] Intermediate vectors cannot replicate themselves in agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and in agrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into agrobacteria (Holsters et al., 1978). Theagrobacterium used as host cell is to comprise a plasmid carrying a virregion. The vir region is necessary for the transfer of the T-DNA intothe plant cell. Additional t-DNA may be contained. The bacterium sotransformed is used for the transformation of plant cells. Plantexplants can advantageously be cultivated with Agrobacterium tumefaciensor Agrobacterium rhizogenes for the transfer of the DNA into the plantcell. Whole plants can then be regenerated from the infected plantmaterial (for example, pieces of leaf, segments of stalk, roots, butalso protoplasts or suspension-cultivated cells) in a suitable medium,which may contain antibiotics or biocides for selection. The plants soobtained can then be tested for the presence of the inserted DNA. Nospecial demands are made of the plasmids in the case of injection andelectroporation. It is possible to use ordinary plasmids, such as, forexample, pUC derivatives. If, for example, the Ti or Ri plasmid is usedfor the transformation of the plant cell, then at least the rightborder, but often the right and the left border of the Ti or Ri plasmidT-DNA, has to be joined as the flanking region of the genes to beinserted. The use of T-DNA for the transformation of plant cells hasbeen intensively researched and sufficiently described in Eur. Pat.Appl. No. EP 120 516; Hockema (1985); An et al., 1985, Herrera-Estrellaet al., (1983), Bevan et al., (1983), and Klee et al., (1985).

[0287] A particularly useful Ti plasmid cassette vector fortransformation of dicotyledonous plants consists of the enhanced CaMV35Spromoter (EN35S) and the 3′ end including polyadenylation signals from asoybean gene encoding the α′-subunit of β-conglycinin. Between these twoelements is a multilinker containing multiple restriction sites for theinsertion of genes of interest.

[0288] The vector preferably contains a segment of pBR322 which providesan origin of replication in E. coli and a region for homologousrecombination with the disarmed T-DNA in Agrobacterium strain ACO; theoriV region from the broad host range plasmid RK1; thestreptomycin/spectinomycin resistance gene from Tn7; and a chimericNPTII gene, containing the CaMV35S promoter and the nopaline synthase(NOS) 3′ end, which provides kanamycin resistance in transformed plantcells.

[0289] Optionally, the enhanced CaMV35S promoter may be replaced withthe 1.5 kb mannopine synthase (MAS) promoter (Velten et al., 1984).After incorporation of a DNA construct into the vector, it is introducedinto A. tumefaciens strain ACO which contains a disarmed Ti plasmid.Cointegrate Ti plasmid vectors are selected and subsequentially may beused to transform a dicotyledonous plant.

[0290]A. tumefaciens ACO is a disarmed strain similar to pTiB6SEdescribed by Fraley et al. (1985). For construction of ACO the startingAgrobacterium strain was the strain A208 which contains a nopaline-typeTi plasmid. The Ti plasmid was disarmed in a manner similar to thatdescribed by Fraley et al. (1985) so that essentially all of the nativeT-DNA was removed except for the left border and a few hundred basepairs of T-DNA inside the left border. The remainder of the T-DNAextending to a point just beyond the right border was replaced with anovel piece of DNA including (from left to right) a segment of pBR322,the oriV region from plasmid RK2, and the kanamycin resistance gene fromTn601. The pBR322 and oriV segments are similar to these segments andprovide a region of homology for cointegrate formation.

[0291] Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

[0292] 4.11.1 Electroporation

[0293] The application of brief, high-voltage electric pulses to avariety of animal and plant cells leads to the formation ofnanometer-sized pores in the plasma membrane. DNA is taken directly intothe cell cytoplasm either through these pores or as a consequence of theredistribution of membrane components that accompanies closure of thepores. Electroporation can be extremely efficient and can be used bothfor transient expression of clones genes and for establishment of celllines that carry integrated copies of the gene of interest.Electroporation, in contrast to calcium phosphate-mediated transfectionand protoplast fusion, frequently gives rise to cell lines that carryone, or at most a few, integrated copies of the foreign DNA.

[0294] The introduction of DNA by means of electroporation, iswell-known to those of skill in the art. In this method, certain cellwall-degrading enzymes, such as pectin-degrading enzymes, are employedto render the target recipient cells more susceptible to transformationby electroporation than untreated cells. Alternatively, recipient cellsare made more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

[0295] 4.11.2 Microprojectile Bombardment

[0296] A further advantageous method for delivering transforming DNAsegments to plant cells is microprojectile bombardment. In this method,particles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

[0297] An advantage of microprojectile bombardment, in addition to itbeing an effective means of reproducibly stably transforming monocots,is that neither the isolation of protoplasts (Cristou et al., 1988) northe susceptibility to Agrobacterium infection is required. Anillustrative embodiment of a method for delivering DNA into maize cellsby acceleration is a Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a filter surface covered withcorn cells cultured in suspension. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing damage inflicted on the recipient cells by projectiles that aretoo large.

[0298] For the bombardment, cells in suspension are preferablyconcentrated on filters or solid culture medium. Alternatively, immatureembryos or other target cells may be arranged on solid culture medium.The cells to be bombarded are positioned at an appropriate distancebelow the macroprojectile stopping plate. If desired, one or morescreens are also positioned between the acceleration device and thecells to be bombarded. Through the use of techniques set forth hereinone may obtain up to 1000 or more foci of cells transiently expressing amarker gene. The number of cells in a focus which express the exogenousgene product 48 hours post-bombardment often range from 1 to 10 andaverage 1 to 3.

[0299] In bombardment transformation, one may optimize theprebombardment culturing conditions and the bombardment parameters toyield the maximum numbers of stable transformants. Both the physical andbiological parameters for bombardment are important in this technology.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment, and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmids. It is believed that pre-bombardment manipulationsare especially important for successful transformation of immatureembryos.

[0300] Accordingly, it is contemplated that one may wish to adjustvarious of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors(TRFs) by modifying conditions which influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

[0301] 4.11.3 Agrobacterium-Mediated Transfer

[0302] Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNAis a relatively precise process resulting in few rearrangements. Theregion of DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., 1986; Jorgensen et al., 1987).

[0303] Modem Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

[0304] Agrobacterium-mediated transformation of leaf disks and othertissues such as cotyledons and hypocotyls appears to be limited toplants that Agrobacterium naturally infects. Agrobacterium-mediatedtransformation is most efficient in dicotyledonous plants. Few monocotsappear to be natural hosts for Agrobacterium, although transgenic plantshave been produced in asparagus using Agrobacterium vectors as described(Bytebier et al., 1987). Therefore, commercially important cereal grainssuch as rice, corn, and wheat must usually be transformed usingalternative methods. However, as mentioned above, the transformation ofasparagus using Agrobacterium can also be achieved (see, for example,Bytebier et al., 1987).

[0305] A transgenic plant formed using Agrobacterium transformationmethods typically contains a single gene on one chromosome. Suchtransgenic plants can be referred to as being heterozygous for the addedgene. However, inasmuch as use of the word “heterozygous” usuallyimplies the presence of a complementary gene at the same locus of thesecond chromosome of a pair of chromosomes, and there is no such gene ina plant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

[0306] More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants produced for enhanced carboxylase activity relativeto a control (native, non-transgenic) or an independent segreganttransgenic plant.

[0307] It is to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes thatencode a polypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

[0308] Transformation of plant protoplasts can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

[0309] Application of these systems to different plant strains dependsupon the ability to regenerate that particular plant strain fromprotoplasts. Illustrative methods for the regeneration of cereals fromprotoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986;Yamada et al., 1986; Abdullah et al., 1986).

[0310] To transform plant strains that cannot be successfullyregenerated from protoplasts, other ways to introduce DNA into intactcells or tissues can be utilized. For example, regeneration of cerealsfrom immature embryos or explants can be effected as described (Vasil,1988). In addition, “particle gun” or high-velocity microprojectiletechnology can be utilized (Vasil, 1992).

[0311] Using that latter technology, DNA is carried through the cellwall and into the cytoplasm on the surface of small metal particles asdescribed (Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988).The metal particles penetrate through several layers of cells and thusallow the transformation of cells within tissue explants.

[0312] 4.11.4 Gene Expression in Plants

[0313] Although great progress has been made in recent years withrespect to preparation of transgenic plants which express bacterialproteins such as B. thuringiensis crystal proteins, the results ofexpressing native bacterial genes in plants are often disappointing.Unlike microbial genetics, little was known by early plant geneticistsabout the factors which affected heterologous expression of foreigngenes in plants. In recent years, however, several potential factorshave been implicated as responsible in varying degrees for the level ofprotein expression from a particular coding sequence. For example,scientists now know that maintaining a significant level of a particularmRNA in the cell is indeed a critical factor. Unfortunately, the causesfor low steady state levels of mRNA encoding foreign proteins are many.First, full length RNA synthesis may not occur at a high frequency. Thiscould, for example, be caused by the premature termination of RNA duringtranscription or due to unexpected mRNA processing during transcription.Second, full length RNA may be produced in the plant cell, but thenprocessed (splicing, polyA addition) in the nucleus in a fashion thatcreates a nonfunctional mRNA. If the RNA is not properly synthesized,terminated and polyadenylated; it cannot move to the cytoplasm fortranslation. Similarly, in the cytoplasm, if mRNAs have reduced halflives (which are determined by their primary or secondary sequence)insufficient protein product will be produced. In addition, there is aneffect, whose magnitude is uncertain, of translational efficiency onmRNA half-life. In addition, every RNA molecule folds into a particularstructure, or perhaps family of structures, which is determined by itssequence. The particular structure of any RNA might lead to greater orlesser stability in the cytoplasm. Structure per se is probably also adeterminant of mRNA processing in the nucleus. Unfortunately, it isimpossible to predict, and nearly impossible to determine, the structureof any RNA (except for tRNA) in vitro or in vivo. However, it is likelythat dramatically changing the sequence of an RNA will have a largeeffect on its folded structure It is likely that structure per se orparticular structural features also have a role in determining RNAstability.

[0314] To overcome these limitations in foreign gene expression,researchers have identified particular sequences and signals in RNAsthat have the potential for having a specific effect on RNA stability.In certain embodiments of the invention, therefore, there is a desire tooptimize expression of the disclosed nucleic acid segments in planta.One particular method of doing so, is by alteration of the bacterialgene to remove sequences or motifs which decrease expression in atransformed plant cell. The process of engineering a coding sequence foroptimal expression in planta is often referred to as “plantizing” a DNAsequence.

[0315] Particularly problematic sequences are those which are A+T rich.Unfortunately, since B. thuringiensis has an A+T rich genome, nativecrystal protein gene sequences must often be modified for optimalexpression in a plant. The sequence motif ATTTA (or AUUUA as it appearsin RNA) has been implicated as a destabilizing sequence in mammaliancell mRNA (Shaw and Kamen, 1986). Many short lived mRNAs have A+T rich3′ untranslated regions, and these regions often have the ATTTAsequence, sometimes present in multiple copies or as multimers (e.g.,ATTTATTTA . . . ). Shaw and Kamen showed that the transfer of the 3′ endof an unstable mRNA to a stable RNA (globin or VA1) decreased the stableRNA's half life dramatically. They further showed that a pentamer ofATTTA had a profound destabilizing effect on a stable message, and thatthis signal could exert its effect whether it was located at the 3′ endor within the coding sequence. However, the number of ATTTA sequencesand/or the sequence context in which they occur also appear to beimportant in determining whether they function as destabilizingsequences. Shaw and Kamen showed that a trimer of ATTTA had much lesseffect than a pentamer on mRNA stability and a dimer or a monomer had noeffect on stability (Shaw and Kamen, 1987). Note that multimers of ATTTAsuch as a pentamer automatically create an A+T rich region. This wasshown to be a cytoplasmic effect, not nuclear. In other unstable mRNAs,the ATTTA sequence may be present in only a single copy, but it is oftencontained in an A+T rich region. From the animal cell data collected todate, it appears that ATTTA at least in some contexts is important instability, but it is not yet possible to predict which occurrences ofATTTA are destabiling elements or whether any of these effects arelikely to be seen in plants.

[0316] Some studies on mRNA degradation in animal cells also indicatethat RNA degradation may begin in some cases with nucleolytic attack inA+T rich regions. It is not clear if these cleavages occur at ATTTAsequences. There are also examples of mRNAs that have differentialstability depending on the cell type in which they are expressed or onthe stage within the cell cycle at which they are expressed. Forexample, histone mRNAs are stable during DNA synthesis but unstable ifDNA synthesis is disrupted. The 3′ end of some histone mRNAs seems to beresponsible for this effect (Pandey and Marzluff, 1987). It does notappear to be mediated by ATTTA, nor is it clear what controls thedifferential stability of this mRNA. Another example is the differentialstability of IgG mRNA in B lymphocytes during B cell maturation(Genovese and Milcarek, 1988). A final example is the instability of amutant β-thallesemic globin mRNA. In bone marrow cells, where this geneis normally expressed, the mutant mRNA is unstable, while the wild-typemRNA is stable. When the mutant gene is expressed in HeLa or L cells invitro, the mutant mRNA shows no instability (Lim et al., 1988). Theseexamples all provide evidence that mRNA stability can be mediated bycell type or cell cycle specific factors. Furthermore this type ofinstability is not yet associated with specific sequences. Given theseuncertainties, it is not possible to predict which RNAs are likely to beunstable in a given cell. In addition, even the ATTTA motif may actdifferentially depending on the nature of the cell in which the RNA ispresent. Shaw and Kamen (1987) have reported that activation of proteinkinase C can block degradation mediated by ATTTA.

[0317] The addition of a polyadenylate string to the 3′ end is common tomost eukaryotic mRNAs, both plant and animal. The currently acceptedview of polyA addition is that the nascent transcript extends beyond themature 3′ terminus. Contained within this transcript are signals forpolyadenylation and proper 3′ end formation. This processing at the 3′end involves cleavage of the mRNA and addition of polyA to the mature 3′end. By searching for consensus sequences near the polyA tract in bothplant and animal mRNAs, it has been possible to identify consensussequences that apparently are involved in polyA addition and 3′ endcleavage. The same consensus sequences seem to be important to both ofthese processes. These signals are typically a variation on the sequenceAATAAA. In animal cells, some variants of this sequence that arefunctional have been identified; in plant cells there seems to be anextended range of functional sequences (Wickens and Stephenson, 1984;Dean et al., 1986). Because all of these consensus sequences arevariations on AATAAA, they all are A+T rich sequences. This sequence istypically found 15 to 20 bp before the polyA tract in a mature mRNA.Studies in animal cells indicate that this sequence is involved in bothpolyA addition and 3′ maturation. Site directed mutations in thissequence can disrupt these functions (Conway and Wickens, 1988; Wickenset al., 1987). However, it has also been observed that sequences up to50 to 100 bp 3′ to the putative polyA signal are also required; i.e., agene that has a normal AATAAA but has been replaced or disrupteddownstream does not get properly polyadenylated (Gil and Proudfoot,1984; Sadofsky and Alwine, 1984; McDevitt et al., 1984). That is, thepolyA signal itself is not sufficient for complete and properprocessing. It is not yet known what specific downstream sequences arerequired in addition to the polyA signal, or if there is a specificsequence that has this function. Therefore, sequence analysis can onlyidentify potential polyA signals.

[0318] In naturally occurring mRNAs that are normally polyadenylated, ithas been observed that disruption of this process, either by alteringthe polyA signal or other sequences in the mRNA, profound effects can beobtained in the level of functional mRNA. This has been observed inseveral naturally occurring mRNAs, with results that are gene-specificso far.

[0319] It has been shown that in natural mRNAs proper polyadenylation isimportant in mRNA accumulation, and that disruption of this process caneffect mRNA levels significantly. However, insufficient knowledge existsto predict the effect of changes in a normal gene. In a heterologousgene, it is even harder to predict the consequences. However, it ispossible that the putative sites identified are dysfunctional. That is,these sites may not act as proper polyA sites, but instead function asaberrant sites that give rise to unstable mRNAs.

[0320] In animal cell systems, AATAAA is by far the most common signalidentified in mRNAs upstream of the polyA, but at least four variantshave also been found (Wickens and Stephenson, 1984). In plants, notnearly so much analysis has been done, but it is clear that multiplesequences similar to AATAAA can be used. The plant sites in Table 5called major or minor refer only to the study of Dean et al. (1986)which analyzed only three types of plant gene. The designation ofpolyadenylation sites as major or minor refers only to the frequency oftheir occurrence as functional sites in naturally occurring genes thathave been analyzed. In the case of plants this is a very limiteddatabase. It is hard to predict with any certainty that a sitedesignated major or minor is more or less likely to function partiallyor completely when found in a heterologous gene such as those encodingthe crystal proteins of the present invention. TABLE 5 POLYADENYLATIONSITES IN PLANT GENES PA AATAAA Major consensus site P1A AATAAT Majorplant site P2A AACCAA Minor plant site P3A ATATAA ″ P4A AATCAA ″ P5AATACTA ″ P6A ATAAAA ″ P7A ATGAAA ″ P8A AAGCAT ″ P9A ATTAAT ″ P10A ATACAT″ P11A AAAATA ″ P12A ATTAAA Minor animal site P13A AATTAA ″ P14A AATACA″ P15A CATAAA ″

[0321] The present invention provides a method for preparing syntheticplant genes which genes express their protein product at levelssignificantly higher than the wild-type genes which were commonlyemployed in plant transformation heretofore. In another aspect, thepresent invention also provides novel synthetic plant genes which encodenon-plant proteins.

[0322] As described above, the expression of native B. thuringiensisgenes in plants is often problematic. The nature of the coding sequencesof B. thuringiensis genes distinguishes them from plant genes as well asmany other heterologous genes expressed in plants. In particular, B.thuringiensis genes are very rich (62%) in adenine (A) and thymine (T)while plant genes and most other bacterial genes which have beenexpressed in plants are on the order of 45-55% A+T.

[0323] Due to the degeneracy of the genetic code and the limited numberof codon choices for any amino acid, most of the “excess” A+T of thestructural coding sequences of some Bacillus species are found in thethird position of the codons. That is, genes of some Bacillus specieshave A or T as the third nucleotide in many codons. Thus A+T content inpart can determine codon usage bias. In addition, it is clear that genesevolve for maximum function in the organism in which they evolve. Thismeans that particular nucleotide sequences found in a gene from oneorganism, where they may play no role except to code for a particularstretch of amino acids, have the potential to be recognized as genecontrol elements in another organism (such as transcriptional promotersor terminators, polyA addition sites, intron splice sites, or specificmRNA degradation signals). It is perhaps surprising that such misreadsignals are not a more common feature of heterologous gene expression,but this can be explained in part by the relatively homogeneous A+Tcontent (˜50%) of many organisms. This A+T content plus the nature ofthe genetic code put clear constraints on the likelihood of occurrenceof any particular oligonucleotide sequence. Thus, a gene from E. coliwith a 50% A+T content is much less likely to contain any particular A+Trich segment than a gene from B. thuringiensis.

[0324] Typically, to obtain high-level expression of the S-endotoxingenes in plants, existing structural coding sequence (“structural gene”)which codes for the S-endotoxin are modified by removal of ATTTAsequences and putative polyadenylation signals by site directedmutagenesis of the DNA comprising the structural gene. It is mostpreferred that substantially all the polyadenylation signals and ATTTAsequences are removed although enhanced expression levels are observedwith only partial removal of either of the above identified sequences.Alternately if a synthetic gene is prepared which codes for theexpression of the subject protein, codons are selected to avoid the AMAsequence and putative polyadenylation signals. For purposes of thepresent invention putative polyadenylation signals include, but are notnecessarily limited to, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA,ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACAand CATAAA. In replacing the ATTTA sequences and polyadenylationsignals, codons are preferably utilized which avoid the codons which arerarely found in plant genomes.

[0325] The selected DNA sequence is scanned to identify regions withgreater than four consecutive adenine (A) or thymine (T) nucleotides.The A+T regions are scanned for potential plant polyadenylation signals.Although the absence of five or more consecutive A or T nucleotideseliminates most plant polyadenylation signals, if there are more thanone of the minor polyadenylation signals identified within tennucleotides of each other, then the nucleotide sequence of this regionis preferably altered to remove these signals while maintaining theoriginal encoded amino acid sequence.

[0326] The second step is to consider the about 15 to about 30 or sonucleotide residues surrounding the A+T rich region identified in stepone. If the A+T content of the surrounding region is less than 80%, theregion should be examined for polyadenylation signals. Alteration of theregion based on polyadenylation signals is dependent upon (1) the numberof polyadenylation signals present and (2) presence of a major plantpolyadenylation signal.

[0327] The extended region is examined for the presence of plantpolyadenylation signals. The polyadenylation signals are removed bysite-directed mutagenesis of the DNA sequence. The extended region isalso examined for multiple copies of the ATTTA sequence which are alsoremoved by mutagenesis.

[0328] It is also preferred that regions comprising many consecutive A+Tbases or G+C bases are disrupted since these regions are predicted tohave a higher likelihood to form hairpin structure due toself-complementarity. Therefore, insertion of heterogeneous base pairswould reduce the likelihood of self-complementary secondary structureformation which are known to inhibit transcription and/or translation insome organisms. In most cases, the adverse effects may be minimized byusing sequences which do not contain more than five consecutive A+T orG+C.

[0329] 4.11.5 Synthetic Oligonucleotides for Mutagenesis

[0330] When oligonucleotides are used in the mutagenesis, it isdesirable to maintain the proper amino acid sequence and reading frame,without introducing common restriction sites such as BglII, HindIII,SacI, KpnI, EcoRI, NcoI, PstI and SalI into the modified gene. Theserestriction sites are found in poly-linker insertion sites of manycloning vectors. Of course, the introduction of new polyadenylationsignals, ATTTA sequences or consecutive stretches of more than five A+Tor G+C, should also be avoided. The preferred size for theoligonucleotides is about 40 to about 50 bases, but fragments rangingfrom about 18 to about 100 bases have been utilized. In most cases, aminimum of about 5 to about 8 base pairs of homology to the template DNAon both ends of the synthesized fragment are maintained to insure properhybridization of the primer to the template. The oligonucleotides shouldavoid sequences longer than five base pairs A+T or G+C. Codons used inthe replacement of wild-type codons should preferably avoid the TA or CGdoublet wherever possible. Codons are selected from a plant preferredcodon table (such as Table 6 below) so as to avoid codons which arerarely found in plant genomes, and efforts should be made to selectcodons to preferably adjust the G+C content to about 50%. TABLE 6PREFERRED CODON USAGE IN PLANTS Percent Usage Amino Acid Codon in PlantsARG CGA 7 CGC 11 CGG 5 CGU 25 AGA 29 AGG 23 LEU CUA 8 CUC 20 CUG 10 CUU28 UUA 5 UUG 30 SER UCA 14 UCC 26 UCG 3 UCU 21 AGC 21 AGU 15 THR ACA 21ACC 41 ACG 7 ACU 31 PRO CCA 45 CCC 19 CCG 9 CCU 26 ALA GCA 23 GCC 32 GCG3 GCU 41 GLY GGA 32 GGC 20 GGG 11 GGU 37 ILE AUA 12 AUC 45 AUU 43 VALGUA 9 GUC 20 GUG 28 GUU 43 LYS AAA 36 AAG 64 ASN AAC 72 AAU 28 GLN CAA64 CAG 36 HIS CAC 65 CAU 35 GLU GAA 48 GAG 52 ASP GAC 48 GAU 52 TYR UAC68 UAU 32 CYS UGC 78 UGU 22 PHE UUC 56 UUU 44 MET AUG 100 TRP UGG 100

[0331] Regions with many consecutive A+T bases or G+C bases arepredicted to have a higher likelihood to form hairpin structures due toself-complementarity. Disruption of these regions by the insertion ofheterogeneous base pairs is preferred and should reduce the likelihoodof the formation of self-complementary secondary structures such ashairpins which are known in some organisms to inhibit transcription(transcriptional terminators) and translation (attenuators).

[0332] Alternatively, a completely synthetic gene for a given amino acidsequence can be prepared, with regions of five or more consecutive A+Tor G+C nucleotides being avoided. Codons are selected avoiding the TAand CG doublets in codons whenever possible. Codon usage can benormalized against a plant preferred codon usage table (such as Table 6)and the G+C content preferably adjusted to about 50%. The resultingsequence should be examined to ensure that there are minimal putativeplant polyadenylation signals and ATTTA sequences. Restriction sitesfound in commonly used cloning vectors are also preferably avoided.However, placement of several unique restriction sites throughout thegene is useful for analysis of gene expression or construction of genevariants.

[0333] 4.11.6 “Plantized” Gene Constructs

[0334] The expression of a plant gene which exists in double-strandedDNA form involves transcription of messenger RNA (mRNA) from one strandof the DNA by RNA polymerase enzyme, and the subsequent processing ofthe mRNA primary transcript inside the nucleus. This processing involvesa 3′ non-translated region which adds polyadenylate nucleotides to the3′ end of the RNA. Transcription of DNA into mRNA is regulated by aregion of DNA usually referred to as the “promoter.” The promoter regioncontains a sequence of bases that signals RNA polymerase to associatewith the DNA and to initiate the transcription of mRNA using one of theDNA strands as a template to make a corresponding strand of RNA.

[0335] A number of promoters which are active in plant cells have beendescribed in the literature. These include the nopaline synthase (NOS)and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of Agrobacterium tumefaciens), the CauliflowerMosaic Virus (CaMV) 19S and 35S promoters, the light-inducible promoterfrom the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO,a very abundant plant polypeptide) and the mannopine synthase (MAS)promoter (Velten et al., 1984 and Velten and Schell, 1985). All of thesepromoters have been used to create various types of DNA constructs whichhave been expressed in plants (see e.g, Int. Pat. Appl. Publ. No. WO84/02913).

[0336] Promoters which are known or are found to cause transcription ofRNA in plant cells can be used in the present invention. Such promotersmay be obtained from plants or plant viruses and include, but are notlimited to, the CaMV35S promoter and promoters isolated from plant genessuch as ssRUBISCO genes. As described below, it is preferred that theparticular promoter selected should be capable of causing sufficientexpression to result in the production of an effective amount ofprotein.

[0337] The promoters used in the DNA constructs (i.e. chimeric plantgenes) of the present invention may be modified, if desired, to affecttheir control characteristics. For example, the CaMV35S promoter may beligated to the portion of the ssRUBISCO gene that represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The resulting chimericpromoter may be used as described herein. For purposes of thisdescription, the phrase “CaMV35S” promoter thus includes variations ofCaMV35S promoter, e.g., promoters derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters may be altered to contain multiple “enhancer sequences” toassist in elevating gene expression.

[0338] The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNA's, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs, as presented in thefollowing examples. Rather, the non-translated leader sequence can bepart of the 5′ end of the non-translated region of the coding sequencefor the virus coat protein, or part of the promoter sequence, or can bederived from an unrelated promoter or coding sequence. In any case, itis preferred that the sequence flanking the initiation site conform tothe translational consensus sequence rules for enhanced translationinitiation reported by Kozak (1984).

[0339] The cry DNA constructs of the present invention may also containone or more modified or fully-synthetic structural coding sequenceswhich have been changed to enhance the performance of the cry gene inplants. The structural genes of the present invention may optionallyencode a fusion protein comprising an amino-terminal chloroplast transitpeptide or secretory signal sequence.

[0340] The DNA construct also contains a 3′ non-translated region. The3′ non-translated region contains a polyadenylation signal whichfunctions in plants to cause the addition of polyadenylate nucleotidesto the 3′ end of the viral RNA. Examples of suitable 3′ regions are (1)the 3′ transcribed, non-translated regions containing thepolyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmidgenes, such as the nopaline synthase (NOS) gene, and (2) plant geneslike the soybean storage protein (7S) genes and the small subunit of theRuBP carboxylase (E9) gene.

[0341] 4.12 Methods for Producting Insect-Resistant Transgenic Plants

[0342] By transforming a suitable host cell, such as a plant cell, witha recombinant cry* gene-containing segment, the expression of theencoded crystal protein (i.e., a bacterial crystal protein orpolypeptide having insecticidal activity against coleopterans) canresult in the formation of insect-resistant plants.

[0343] By way of example, one may utilize an expression vectorcontaining a coding region for a B. thuringiensis crystal protein and anappropriate selectable marker to transform a suspension of embryonicplant cells, such as wheat or corn cells using a method such as particlebombardment (Maddock et al., 1991; Vasil et al., 1992) to deliver theDNA coated on microprojectiles into the recipient cells. Transgenicplants are then regenerated from transformed embryonic calli thatexpress the insecticidal proteins.

[0344] The formation of transgenic plants may also be accomplished usingother methods of cell transformation which are known in the art such asAgrobacterium-mediated DNA transfer (Fraley et al., 1983).Alternatively, DNA can be introduced into plants by direct DNA transferinto pollen (Zhou et al., 1983; Hess, 1987; Luo et al., 1988), byinjection of the DNA into reproductive organs of a plant (Pena et al.,1987), or by direct injection of DNA into the cells of immature embryosfollowed by the rehydration of desiccated embryos (Neuhaus et al., 1987;Benbrook et al., 1986).

[0345] The regeneration, development, and cultivation of plants fromsingle plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, 1988). Thisregeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil.

[0346] The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983).

[0347] This procedure typically produces shoots within two to fourmonths and those shoots are then transferred to an appropriateroot-inducing medium containing the selective agent and an antibiotic toprevent bacterial growth. Shoots that rooted in the presence of theselective agent to form plantlets are then transplanted to soil or othermedia to allow the production of roots. These procedures vary dependingupon the particular plant strain employed, such variations being wellknown in the art.

[0348] Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

[0349] Such plants can form germ cells and transmit the transformedtrait(s) to progeny plants. Likewise, transgenic plants can be grown inthe normal manner and crossed with plants that have the same transformedhereditary factors or other hereditary factors. The resulting hybridindividuals have the corresponding phenotypic properties. A transgenicplant of this invention thus has an increased amount of a coding region(e.g., a mutated cry gene) that encodes the mutated Cry polypeptide ofinterest. A preferred transgenic plant is an independent segregant andcan transmit that gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for that gene, and transmits that gene toall of its offspring on sexual mating.

[0350] Seed from a transgenic plant may be grown in the field orgreenhouse, and resulting sexually mature transgenic plants areself-pollinated to generate true breeding plants. The progeny from theseplants become true breeding lines that are evaluated for, by way ofexample, increased insecticidal capacity against coleopteran insects,preferably in the field, under a range of environmental conditions. Theinventors contemplate that the present invention will find particularutility in the creation of transgenic plants of commercial interestincluding various grasses, grains, fibers, tubers, legumes, ornamentalplants, cacti, succulents, fruits, berries, and vegetables, as well as anumber of nut- and fruit-bearing trees and plants.

[0351] 4.13 Methods for Producing Combinatorial Cry3* Variants

[0352] Crystal protein mutants containing substitutions in one or moredomains may be constructed via a number of techniques. For instance,sequences of highly related genes can be readily shuffled using thePCR™-based technique described by Stemmer (1994). Alternatively, ifsuitable restriction sites are available, the mutations of one cry genemay be combined with the mutations of a second cry gene by routinesubcloning methodologies. If a suitable restriction site is notavailable, one may be generated by oligonucleotide directed mutagenesisusing any number of procedures known to those skilled in the art.Alternatively, splice-overlap extension PCR™ (Horton et al., 1989) maybe used to combine mutations in different regions of a crystal protein.In this procedure, overlapping DNA fragments generated by the PCR™ andcontaining different mutations within their unique sequences may beannealed and used as a template for amplification using flanking primersto generate a hybrid gene sequence. Finally, cry* mutants may becombined by simply using one cry mutant as a template foroligonucleotide-directed mutagenesis using any number of protocols suchas those described herein.

[0353] 4.14 Isolating Homologous Gene and Gene Fragments

[0354] The genes and δ-endotoxins according to the subject inventioninclude not only the full length sequences disclosed herein but alsofragments of these sequences, or fusion proteins, which retain thecharacteristic insecticidal activity of the sequences specificallyexemplified herein.

[0355] It should be apparent to a person skill in this art thatinsecticidal δ-endotoxins can be identified and obtained through severalmeans. The specific genes, or portions thereof, may be obtained from aculture depository, or constructed synthetically, for example, by use ofa gene machine. Variations of these genes may be readily constructedusing standard techniques for making point mutations. Also, fragments ofthese genes can be made using commercially available exonucleases orendonucleases according to standard procedures. For example, enzymessuch as Bal31 or site-directed mutagenesis can be used to systematicallycut off nucleotides from the ends of these genes. Also, genes which codefor active fragments may be obtained using a variety of otherrestriction enzymes. Proteases may be used to directly obtain activefragments of these δ-endotoxins.

[0356] Equivalent δ-endotoxins and/or genes encoding these equivalentδ-endotoxins can also be isolated from Bacillus strains and/or DNAlibraries using the teachings provided herein. For example, antibodiesto the δ-endotoxins disclosed and claimed herein can be used to identifyand isolate other δ-endotoxins from a mixture of proteins. Specifically,antibodies may be raised to the portions of the δ-endotoxins which aremost constant and most distinct from other B. thuringiensisδ-endotoxins. These antibodies can then be used to specifically identifyequivalent δ-endotoxins with the characteristic insecticidal activity byimmunoprecipitation, enzyme linked immunoassay (ELISA), or Westernblotting.

[0357] A further method for identifying the δ-endotoxins and genes ofthe subject invention is through the use of oligonucleotide probes.These probes are nucleotide sequences having a detectable label. As iswell known in the art, if the probe molecule and nucleic acid samplehybridize by forming a strong bond between the two molecules, it can bereasonably assumed that the probe and sample are essentially identical.The probe's detectable label provides a means for determining in a knownmanner whether hybridization has occurred. Such a probe analysisprovides a rapid method for identifying formicidal δ-endotoxin genes ofthe subject invention.

[0358] The nucleotide segments which are used as probes according to theinvention can be synthesized by use of DNA synthesizers using standardprocedures. In the use of the nucleotide segments as probes, theparticular probe is labeled with any suitable label known to thoseskilled in the art, including radioactive and non-radioactive labels.Typical radioactive labels include ³²P, ¹²⁵I, ³⁵S, or the like. A probelabeled with a radioactive isotope can be constructed from a nucleotidesequence complementary to the DNA sample by a conventional nicktranslation reaction, using a DNase and DNA polymerase. The probe andsample can then be combined in a hybridization buffer solution and heldat an appropriate temperature until annealing occurs. Thereafter, themembrane is washed free of extraneous materials, leaving the sample andbound probe molecules typically detected and quantified byautoradiography and/or liquid scintillation counting.

[0359] Non-radioactive labels include, for example, ligands such asbiotin or thyroxine, as well as enzymes such as hydrolases orperoxidases, or the various chemiluminescers such as luciferin, orfluorescent compounds like fluorescein and its derivatives. The probemay also be labeled at both ends with different types of labels for easeof separation, as, for example, by using an isotopic label at the endmentioned above and a biotin label at the other end.

[0360] Duplex formation and stability depend on substantialcomplementarity between the two strands of a hybrid, and, as notedabove, a certain degree of mismatch can be tolerated. Therefore, theprobes of the subject invention include mutations (both single andmultiple), deletions, insertions of the described sequences, andcombinations thereof, wherein said mutations, insertions and deletionspermit formation of stable hybrids with the target polynucleotide ofinterest. Mutations, insertions, and deletions can be produced in agiven polynucleotide sequence in many ways, by methods currently knownto an ordinarily skilled artisan, and perhaps by other methods which maybecome known in the future.

[0361] The potential variations in the probes listed is due, in part, tothe redundancy of the genetic code. Because of the redundancy of thegenetic code, i.e., more than one coding nucleotide triplet (codon) canbe used for most of the amino acids used to make proteins. Thereforedifferent nucleotide sequences can code for a particular amino acid.Thus, the amino acid sequences of the B. thuringiensis δ-endotoxins andpeptides can be prepared by equivalent nucleotide sequences encoding thesame amino acid sequence of the protein or peptide. Accordingly, thesubject invention includes such equivalent nucleotide sequences. Also,inverse or complement sequences are an aspect of the subject inventionand can be readily used by a person skilled in this art. In addition ithas been shown that proteins of identified structure and function may beconstructed by changing the amino acid sequence if such changes do notalter the protein secondary structure (Kaiser and Kezdy, 1984). Thus,the subject invention includes mutants of the amino acid sequencedepicted herein which do not alter the protein secondary structure, orif the structure is altered, the biological activity is substantiallyretained. Further, the invention also includes mutants of organismshosting all or part of a δ-endotoxin encoding a gene of the invention.Such mutants can be made by techniques well known to persons skilled inthe art. For example, UV irradiation can be used to prepare mutants ofhost organisms. Likewise, such mutants may include asporogenous hostcells which also can be prepared by procedures well known in the art.

[0362] 4.15 Ribozymes

[0363] Ribozymes are enzymatic RNA molecules which cleave particularmRNA species. In certain embodiments, the inventors contemplate theselection and utilization of ribozymes capable of cleaving the RNAsegments of the present invention, and their use to reduce activity oftarget mRNAs in particular cell types or tissues.

[0364] Six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

[0365] The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

[0366] The enzymatic nucleic acid molecule may be formed in ahammerhead, hairpin, a hepatitis δ virus, group I intron or RNaseP RNA(in association with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992);examples of hairpin motifs are described by Hampel et al. (Eur. Pat. EP0360257), Hampel and Tritz (1989), Hampel et al. (1990) and Cech et al.(U.S. Pat. No. 5,631,359; an example of the hepatitis δ virus motif isdescribed by Perrotta and Been (1992); an example of the RNaseP motif isdescribed by Guerrier-Takada et al. (1983); Neurospora VS RNA ribozymemotif is described by Collins (Saville and Collins, 1990; Saville andCollins, 1991; Collins and Olive, 1993); and an example of the Group Iintron is described by Cech et al. (U.S. Pat. No. 4,987,071). All thatis important in an enzymatic nucleic acid molecule of this invention isthat it has a specific substrate binding site which is complementary toone or more of the target gene RNA regions, and that it have nucleotidesequences within or surrounding that substrate binding site which impartan RNA cleaving activity to the molecule. Thus the ribozyme constructsneed not be limited to specific motifs mentioned herein.

[0367] The invention provides a method for producing a class ofenzymatic cleaving agents which exhibit a high degree of specificity forthe RNA of a desired target. The enzymatic nucleic acid molecule ispreferably targeted to a highly conserved sequence region of a targetmRNA such that specific treatment of a disease or condition can beprovided with either one or several enzymatic nucleic acids. Suchenzymatic nucleic acid molecules can be delivered exogenously tospecific cells as required. Alternatively, the ribozymes can beexpressed from DNA or RNA vectors that are delivered to specific cells.

[0368] Small enzymatic nucleic acid motifs (e.g., of the hammerhead orthe hairpin structure) may be used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzymaticnucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet etal., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (Draper et al., Int. Pat. Appl. Publ. No. WO 93/23569, andSullivan et al., Int. Pat. Appl. Publ. No. WO 94/02595, both herebyincorporated in their totality by reference herein; Ohkawa et al, 1992;Taira et al., 1991; Ventura et al., 1993).

[0369] Ribozymes may be added directly, or can be complexed withcationic lipids, lipid complexes, packaged within liposomes, orotherwise delivered to target cells. The RNA or RNA complexes can belocally administered to relevant tissues ex vivo, or in vivo throughinjection, aerosol inhalation, infusion pump or stent, with or withouttheir incorporation in biopolymers.

[0370] Ribozymes may be designed as described in Draper et al. (Int.Pat. Appl. Publ. No. WO 93/23569), or Sullivan et al., (Int. Pat. Appl.Publ. No. WO 94/02595) and synthesized to be tested in vitro and invivo, as described. Such ribozymes can also be optimized for delivery.While specific examples are provided, those in the art will recognizethat equivalent RNA targets in other species can be utilized whennecessary.

[0371] Hammerhead or hairpin ribozymes may be individually analyzed bycomputer folding (Jaeger et al., 1989) to assess whether the ribozymesequences fold into the appropriate secondary structure. Those ribozymeswith unfavorable intramolecular interactions between the binding armsand the catalytic core are eliminated from consideration. Varyingbinding arm lengths can be chosen to optimize activity. Generally, atleast 5 bases on each arm are able to bind to, or otherwise interactwith, the target RNA.

[0372] Ribozymes of the hammerhead or hairpin motif may be designed toanneal to various sites in the mRNA message, and can be chemicallysynthesized. The method of synthesis used follows the procedure fornormal RNA synthesis as described in Usman et al. (1987) and in Scaringeet al. (1990) and makes use of common nucleic acid protecting andcoupling groups, such as dimethoxytrityl at the 5′-end, andphosphoramidites at the 3′-end. Average stepwise coupling yields aretypically >98%. Hairpin ribozymes may be synthesized in two parts andannealed to reconstruct an active ribozyme (Chowrira and Burke, 1992).Ribozymes may be modified extensively to enhance stability bymodification with nuclease resistant groups, for example, 2′-amino,2′-C-allyl, 2′-flouro, 2′-o-methyl, 2′-H (for a review see Usman andCedergren, 1992). Ribozymes may be purified by gel electrophoresis usinggeneral methods or by high pressure liquid chromatography andresuspended in water.

[0373] Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

[0374] Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describesthe general methods for delivery of enzymatic RNA molecules. Ribozymesmay be administered to cells by a variety of methods known to thosefamiliar to the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) andDraper et al. (Int. Pat. Appl. Publ. No. WO 93/23569) which have beenincorporated by reference herein.

[0375] Another means of accumulating high concentrations of aribozyme(s) within cells is to incorporate the ribozyme-encodingsequences into a DNA expression vector. Transcription of the ribozymesequences are driven from a promoter for eukaryotic RNA polymerase I(pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).Transcripts from pol II or pol III promoters will be expressed at highlevels in all cells; the levels of a given pol II promoter in a givencell type will depend on the nature of the gene regulatory sequences(enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerasepromoters may also be used, providing that the prokaryotic RNApolymerase enzyme is expressed in the appropriate cells (Elroy-Stein andMoss, 1990; Gao and Huang, 1993; Lieber et al., 1993; Zhou et al.,1990). Ribozymes expressed from such promoters can function in mammaliancells (e.g Kashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al.,1992; Yu et al., 1993; L'Huillier et al., 1992; Lisziewicz et al.,1993). Such transcription units can be incorporated into a variety ofvectors for introduction into mammalian cells, including but notrestricted to, plasmid DNA vectors, viral DNA vectors (such asadenovirus or adeno-associated vectors), or viral RNA vectors (such asretroviral, semliki forest virus, sindbis virus vectors).

[0376] Ribozymes of this invention may be used as diagnostic tools toexamine genetic drift and mutations within cell lines or cell types.They can also be used to assess levels of the target RNA molecule. Theclose relationship between ribozyme activity and the structure of thetarget RNA allows the detection of mutations in any region of themolecule which alters the base-pairing and three-dimensional structureof the target RNA. By using multiple ribozymes described in thisinvention, one may map nucleotide changes which are important to RNAstructure and function in vitro, as well as in cells and tissues.Cleavage of target RNAs with ribozymes may be used to inhibit geneexpression and define the role (essentially) of specified gene productsin particular cells or cell types.

[0377] 5.0 EXAMPLES

[0378] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

5.1 Example 1

[0379] Three-Dimensional Structure of Cry3Bb

[0380] The three-dimensional structure of Cry3Bb was determined by X-raycrystallography. Crystallization of Cry3Bb and X-ray diffraction datacollection were performed as described by Cody et al. (1992). Thecrystal structure of Cry3Bb was refined to a residual R factor of 18.0%using data collected to 2.4 Å resolution. The crystals belong to thespace group C222₁ with unit cell dimensions a=122.44, b=131.81, andc=105.37 Å and contain one molecule in the asymmetric unit. Atomiccoordinates for Cry3Bb are described in Example 31 and listed in Section9.

[0381] The structure of Cry3Bb is similar to that of Cry3A (Li et al.,1991). It consists of 5825 protein atoms from 588 residues (amino acids64-652) forming three discrete domains (FIG. 1). A total of 251 watermolecules have been identified in the Cry3Bb structure (FIG. 2). Domain1 (residues 64-294) is a seven helical bundle formed by six helicestwisted around the central helix, α5 (FIG. 3). The amino acids formingeach helix are listed in FIG. 4. Domain 2 (residues 295-502) containsthree antiparallel β-sheets (FIG. 5A and FIG. 5B). Sheets 1 and 2, eachcomposed of 4 β strands, form the distinctive “Greek key” motif. Theouter surface of sheet 3, composed of 3 β strands, makes contact withhelix α7 of domain 1. FIG. 6 lists the amino acids comprising each βstrand in domain 2. A small α helix, α8 which follows β strand 1, isalso included in domain 2. Domain 3 (residues 503-652) has a “jellyroll” β-barrel topology which has a hydrophobic core and is nearlyparallel to the a and perpendicular to the c axes of the lattice (FIG.7A and FIG. 7B). The amino acids comprising each β strand of domain 3are listed in FIG. 8.

[0382] The monomers of Cry3Bb in the crystal form a dimeric quaternarystructure along a two-fold axis parallel to the a axis (FIG. 9A and FIG.9B). Helix α6 lies in a cleft formed by the interface of domain 1 anddomains 1 and 3 of its symmetry related molecule. There are numerousclose hydrogen bonding contacts along this surface, confirming thestructural stability of the dimer.

5.2 Example 2

[0383] Preparation of Cry3Bb.60

[0384]B. thuringiensis EG7231 was grown through sporulation in C2 mediumwith chloramphenicol (Cml) selection. The solids from this culture wererecovered by centrifugation and washed with water. The toxin waspurified by recrystallization from 4.0 M NaBr (Cody et al., 1992). Thepurified Cry3Bb was solubilized in 10 ml of 50 mM KOH/100 mg Cry3Bb andbuffered to pH 9.0 with 100 mM CAPS (pH 9.0). The soluble toxin wastreated with trypsin at a weight ratio of 50 mg toxin to 1 mg trypsin.After 20 min of trypsin digestion the predominant protein visualized bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) was 60 kDa. Furtherdigestion of the 60-kDa toxin was not observed. FIG. 4 illustrates theCoomassie-stained Cry3Bb and Cry3Bb.60 following SDS-PAGE.

5.3 Example 3

[0385] Purification and Sequencing of Cry3Bb.60

[0386] Cry3Bb.60 was electrophoretically purified by SDS-PAGE andelectroblotted to Immobilon-P® (Millipore) membrane by semi-dry transferat 15V for 30 min. The membrane was then washed twice with water andstained with 0.025% R-250, 40% methanol. To reduce the background, theblot was destained with 50% methanol until the stained protein bandswere visible. The blot was then air dried, and the stained Cry3Bb.60band was cut out of the membrane. This band was sent to the TuftsUniversity Sequencing Laboratory (Boston, Mass.) for N-terminalsequencing. The experimentally-determined N-terminal amino acid sequenceis shown in Table 7 beside the known amino acid sequence starting atamino acid residue 160. TABLE 7 AMINO ACID SEQUENCE OF THE N-TERMINUS OFCRY3BB.60 AND COMPARISON TO THE KNOWN SEQUENCE OF CRY3BB Deduced KnownSequence Sequence Residue # S S 160 K K 161 R R 162 S S 163 Q Q 164 D D165 R R 166

5.4 Example 4

[0387] Bioactivity of Cry3Bb.60

[0388] Cry3Bb was prepared for bioassay by solubilization in a minimalamount of 50 mM KOH, 10 ml per 100 mg toxin, and buffered to pH 9.0 with100 mM CAPS, pH 9.0. Cry3Bb.60 was prepared as described in Example 1.Both preparations were kept at room temperature 12 to 16 hours prior tobioassay. After seven days the mortality of the population wasdetermined and analyzed to determine the lethal concentration of eachtoxin. These results are numerized in Table 8. TABLE 8 BIOACTIVITY OFCRY3BB AND CRY3BB.60 AGAINST THE SOUTHERN CORN ROOTWORM (DIABIOTICAUNDECIMPUNCTATA) LC₅₀ mg/well 95% C. I. Cry3Bb 24.09 15-39 Cry3Bb.60 6.72 5.25-8.4 

5.5 Example 5

[0389] Ion-Channel Formation by Cry3Bb and CryB2.60

[0390] Cry3Bb.60 and Cry3Bb were evaluated for their ability to form ionchannels in planar lipid bilayers. Bilayers of phosphatidylcholine wereformed on Teflon® supports over a 0.7-mm hole. A bathing solution of 3.5ml 100 mM KOH, 10 mM CaCl₂, 100 mM CAPS (pH 9.5) was placed on eitherside of the Teflon® partition. The toxin was added to one side of thepartition and a voltage of 60 mV was imposed across thephosphatidylcholine bilayer. Any leakage of ions through the membranewas amplified and recorded. An analysis of the frequency of theconductances created by either Cry3Bb or Cry3Bb.60 are illustrated inFIG. 5A and FIG. 5B. Cry3Bb.60 readily formed ion channels whereasCry3Bb rarely formed channels.

5.6 Example 6

[0391] Formation of High Molecular-Weight Oligomers

[0392] Individual molecules of Cry3Bb or Cry3Bb.60 form a complex withanother like molecule. The ability of Cry3Bb to form an oligomer is notreproducibly apparent. The complex cannot be repeatedly observed to formunder nondenaturing conditions. Cry3Bb.60 formed a significantly greateramount of a higher molecular-weight complex (≧120 kDa) with otherCry3Bb.60 molecules. Oligomers of Cry3Bb are demonstrated by theintensity of the Coomassie-stained SDS polyacrylamide gel.Oligomerization is visualized on SDS-PAGE by not heating samples priorto loading on the gel to retain some nondenatured toxin. These datasuggest that Cry3Bb.60 more readily forms the higher order complex thanCry3Bb alone. Oligomerization is also observed by studying theconductance produced by these molecules and the time-dependent increasein conductance. This change in conductance can be attributed tooligomerization of the toxin.

5.7 Example 7

[0393] Design Method 1: Identification and Alteration ofProtease-Sensitive Sites and Proteolytic Processing

[0394] It has been reported in the literature that treatment of Cry3Atoxin protein with trypsin, an enzyme that cleaves proteins on thecarboxyl side of available lysine and arginine residues, yields a stablecleavage product of 55 kDa from the 67 kDa native protein (Carroll etal., 1989). N-terminal sequencing of the 55 kDa product showed cleavageoccurs at amino acid residue R158. The truncated Cry3A protein was foundto retain the same level of insecticidal activity as the native protein.Cry3Bb toxin protein was also treated with trypsin. After digestion, theprotein size decreased from 68 kDa, the molecular weight of the nativeCry3Bb toxin, to 60 kDa. No further digestion was observed. N-terminalsequencing revealed the trypsin cleavage site of the truncated toxin(Cry3Bb.60) to be amino acid R159 in lα3,4 of Cry3Bb. Unexpectedly, thebioactivity of the truncated Cry3Bb toxin was found to increase.

[0395] Using this method, protease digestion of a B. thuringiensis toxinprotein, a proteolytically sensitive site was identified on Cry3Bb, anda more highly active form of the protein (Cry3Bb.60) was identified.Modifications to this proteolytically-sensitive site by introducing anadditional protease recognition site also resulted in the isolation of abiologically more active protein. It is also possible that removal ofother protease-sensitive site(s) may improve activity. Proteolyticallysensitive regions, once identified, may be modified or utilized toproduce biologically more active toxins.

[0396] 5.7.1 Cry3Bb.60

[0397] Treatment of solubilized Cry3Bb toxin protein with trypsinresults in the isolation of a stable, truncated Cry3Bb toxin proteinwith a molecular weight of 60 kDa (Cry3Bb.60). N-terminal sequencing ofCry3Bb.60 shows the trypsin-sensitive site to be R159 in lα3,4 of thenative toxin. Trypsin digestion results in the removal of helices 1-3from the native Cry3Bb but also increases the activity of the toxinagainst SCRW larvae approximately four-fold.

[0398] Cry3Bb.60 is a unique toxin with enhanced insecticidal use overthe parent Cry3Bb. Improved biological activity, is only one parameterthat distinguishes it as a new toxin. Aside from the reduced size,Cry3Bb.60 is also a more soluble protein. Cry3Bb precipitates fromsolution at pH 6.5 while Cry3Bb.60 remains in solution from pH 4.5 to pH12. Cry3Bb.60 also forms ion channels with greater frequency thanCry3Bb.

[0399] Cry3Bb.60 is produced by either the proteolytic removal of thefirst 159 amino acid residues, or the in vivo production of this toxin,by bacteria or plants expressing the gene for Cry3Bb.60, that is, theCry3Bb gene without the first 483 nucleotides.

[0400] In conclusion, Cry3Bb.60 is distinct from Cry3Bb in severalimportant ways: enhanced insecticidal activity; enhanced range ofsolubility; enhanced ability to form channels; and reduced size.

[0401] 5.7.2 EG11221

[0402] Semi-random mutagenesis of the trypsin-sensitive lα3,4 region ofCry3Bb resulted in the isolation of Cry3Bb.11221, a designed Cry3Bbprotein that exhibits over a 6-fold increase in activity against SCRWlarvae compared to WT. Cry3Bb.11221 has 4 amino acid changes in thelα3,4 region. One of these changes, L158R, introduces an additionaltrypsin site adjacent to R159, the proteolytically sensitive site usedto produce Cry3Bb.60 (example 4.1.1). Cry3Bb.11221 is produced by B.thuringiensis as a full length toxin protein but is presumably digestedby insect gut proteases to the same size as Cry3Bb.60 (see Cry3A resultsfrom Carroll et al., 1989). The additional protease recognition site maymake the lα3,4 region even more sensitive to digestion, therebyincreasing activity.

5.8 Example 8

[0403] Design Method 2: Determining and Manipulation of Bound Water

[0404] There are several ways that water molecules can associate with aprotein, including surface water that is easily removed and bound waterthat is more difficult to extract (Dunitz, 1994; Zhang and Matthews,1994). The function of bound water has been the subject of significantacademic extrapolation, but the precise function has little experimentalvalidation. Some of the most interesting bound or structural water isthe water that participates in the protein structure from inside theprotein itself.

[0405] The occupation of a site by a water molecule can indicate astable pocket within a protein or a looseness of packing created bywater-mediated salt bridges and hydrogen bonding to water. This canreduce the degree of bonding between amino acids, possibly making theregion more flexible. A different amino acid sequence around that samesite could result in better packing, collapsing the pocket around polaror charged amino acids. This may result in decreased flexibility.Therefore, the degree of hydration of a region of a protein maydetermine the flexibility or mobility of that region, and manipulationof the hydration may alter the flexibility. Methods of increasing thehydration of a water-exposed region include increasing the number ofhydrophobic residues along that surface. It is taught in the art thatexposed hydrophobic residues require significantly more water to hydratethan hydrophilic residues (CRC Handbook of Chemistry and Physics, CRCPress, Inc.). It is not taught, however, that by doing this,improvements to the biological activity of a protein can be achieved.

[0406] Structural water has not previously been identified in B.thuringiensis δ-endotoxins including Cry3Bb. Furthermore, there are noreports of the function of this structural water in δ-endotoxins orbacterial toxins. In the analysis of Cry3Bb, it was observed that acollection of water molecules are located around lα3,4, a site definedby the inventors as important for improvement of bioactivity. The loopα3,4 region is surface exposed and may define a hinge in the proteinpermitting either removal or movement of the first three helices ofdomain 1. The hydration found around this region may impart flexibilityand mobility to this loop. The observation of structural water at thelα3,4 site provided an analytical tool for further structure analysis.If this important site is surrounded by water, then other importantsites may also be completely or partially surrounded by water. Usingthis insight, structural water surrounding helices 5 and 6 was thenidentified. This structural water forms a column through the protein,effectively separating helices 5 and 6 from the rest of the molecule.The structures of Cry3A and Cry3Bb suggest that helices 5 and 6 aretightly associated, bound together by Van der Waals interactions. Alone,helix 5 from Cry3A, although insufficient for biological activity, hasbeen demonstrated to have the ability to form ion channels in anartificial membrane (Gazit and Shai, 1993). The ion channels formed byhelix 5 are 10-fold smaller than the channels of the full length toxinsuggesting that significantly more toxin structure is required for thefull-sized ion channels. In Cry3Bb, helix 5 as part of a cluster of αhelices (domain 1) has been found to form ion channels (Von Tersch etal., 1994). Unpublished experimental observations by the inventorsdemonstrate that helix 6 also crossed the biological membrane. Helices 5and 6, therefore, are the putative channel-forming helices necessary fortoxicity.

[0407] The hydration around these helices may indicate that flexibilityof this region is necessary for toxicity. It is conceivable, therefore,that if it were possible to improve the hydration around helices 5 and6, one could create a better toxin protein. Care must be taken, however,to avoid creating continuous hydrophobic surfaces between helices 5-6and any other part of the protein which could, by hydrophobicinteractions, act to restrict movement of the mobile helices. Themobility of helices 5 and 6 may also depend on the flexibility of theloops attached to them as well as on other regions of the Cry3Bbmolecule, particularly in domain 1, which may undergo conformationalchanges to allow insertion of the 2 helices into the membrane. Alteringthe hydration of these regions of the protein may also affect itsbioactivity.

[0408] 5.8.1 Cry3Bb.11032

[0409] A collection of bound water residues indicated the relativeflexibility of the lα3,4 region. The flexibility of this loop can beincreased by increasing the hydration of the region by substitutingrelatively hydrophobic residues for the exposed hydrophilic residues. Anexample of an improved, designed protein having this type ofsubstitution is Cry3Bb.11032. Cry3Bb.11032 has the amino acid changeD165G; glycine is more hydrophobic than aspartate (Kyte and Doolittlehydrophobicity score of −0.4 vs. −3.5 for aspartate). Cry3Bb.11032 isapproximately 3 times more active than WT Cry3Bb.

[0410] 5.8.2 Cry3Bb.11051

[0411] To increase the hydration of the lα4,5 region of Cry3Bb, glycinewas substituted for the surface exposed residue K189. Glycine is morehydrophobic than lysine (Kyte and Doolittle hydrophobicity score of −0.4vs. −3.9 for lysine) and may result in an increase in bound water. Theincrease in bound water may impart greater flexibility to the loopregion which precedes the channel-forming helix, α5. The designed Cry3Bbprotein with the K189G change, Cry3Bb.11051, exhibits a 3-fold increasein activity compared to WT Cry3Bb.

[0412] 5.8.3 Alterations to Lα7,β1 (Cry3Bb.11241 and 11242)

[0413] Amino acid changes made in the surface-exposed loop connectingα-helix 7 and β-strand 1 (lα7,β1) resulted in the identification of 2altered Cry3Bb proteins with increased bioactivities, Cry3Bb.11241 andCry3Bb.11242. Analysis of the hydropathy index of 2 of these proteinsover the 20 amino acid sequence 281-300, inclusive of the lα7,β1 region,reveal that the amino acid substitutions in these proteins have made thelα7,β1 region much more hydrophobic. The grand average of hydropathyvalue (GRAVY) was determined for each protein sequence using thePC\GENE® (IntelliGenetics, Inc., Mountain View, Calif., release 6.85)protein sequence analysis computer program, SOAP, and a 7 amino acidinterval. The SOAP program is based on the method of Kyte and Doolittle(1982). The increase in hydrophobicity of the lα7,β1 region for eachprotein may increase the hydration of the loop and, therefore, theflexibility. The altered proteins, their respective amino acid changes,fold-increases over WT bioactivity, and GRAVY values are listed in Table9. TABLE 9 HYDROPATHY VALUES FOR THE Lα7, β1 REGION OF CRY3BB AND 2DESIGNED CRY3BB PROTEINS SHOWING INCREASED SCRW BIOACTIVITY Amino FoldIncrease in Cry3Bb* Acid Bioactivity Over GRAVY Protein Changes WT(Amino Acids 281-300) wildtype — — 4.50 Cry3Bb.11241 Y287F, 2.6× 10.70D288N, R290L Cry3Bb.11242 R290V 2.5× 8.85

[0414] 5.8.4 Alterations to Lβ1,α8 (Cry3Bb.11228, Cry3Bb.11229,Cry3Bb.11230, Cry3Bb.11233, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238 andCry3Bb.11239)

[0415] The surface-exposed loop between β-strand 1 and α-helix 8(lβ1,α8) defines the boundary between domains 1 and 2 of Cry3Bb. Theintroduction of semi-random amino acid changes to this region resultedin the identification of several altered Cry3Bb proteins with increasedbioactivity. Hydropathy index analysis of the amino acid substitutionsfound in the altered proteins shows that the changes have made theexposed region more hydrophobic which may result in increased hydrationand flexibility. Table 10 lists the altered proteins, their respectiveamino acid changes and fold increases over WT Cry3Bb and the grandaverage of hydropathy value (GRAVY) determined using the PC\GENE®(IntelliGenetics, Inc., Mountain View, Calif., release 6.85) proteinsequence analysis prograrn, SOAP, over the 20 amino acid sequence305-324 inclusive of lβ1,α8 using a 7 amino acid interval. TABLE 10HYDROPATHY VALUES FOR THE Lβ1, α8 REGION OF CRY3BB AND 8 DESIGNEDCRY3BB* PROTEINS SHOWING INCREASED SCRW BIOACTIVITY Fold Increase inGRAVY Cry3Bb* Amino Acid Bioactivity Over (Amino Acids Protein ChangesWild Type 305-324) wildtype — — 0.85 Cry3Bb.11228 S311L, N313T, 4.1×4.35 E317K Cry3Bb.11229 S311T, E317K, 2.5× 2.60 Y318C Cry3Bb.11230S311A, L312V, 4.7× 3.65 Q316W Cry3Bb.11233 S311A, Q316D 2.2× 2.15Cry3Bb.11236 S311I 3.1× 3.50 Cry3Bb.11237 S311I, N313H 5.4× 3.65Cry3Bb.11238 N313V, T314N, 2.6× 9.85 Q316M, E317V Cry3Bb.11239 N313R,L315P, 2.8× 3.95 Q316L, E317A

[0416] 5.8.5 Cry3Bb.11227, Cry3Bb.11241 and Cry3Bb.11242

[0417] Amino acid Q238, located in helix 6 of Cry3Bb, has beenidentified as a residue that, by its large size and hydrogen bonding toR290, blocks complete hydration of the space between helix 6 and helix4. Substitution of R290 with amino acids that do not form hydrogen bondsor that have side chains that can not span the physical distance tohydrogen bond with Q238 may result in increased hydration around Q238.Q238, unable to hydrogen bond to R290, may now bind water. This mayincrease the flexibility of the channel-forming region. Designedproteins Cry3Bb.11227 (R290N), Cry3Bb.11241 (R290L) and Cry3Bb.11242(R290V) show increased activities of approximately 2-fold, 2.6-fold and2.5-fold, respectively, against SCRW larvae compared to WT.

5.9 Example 9

[0418] Design Method 3: Manipulation of Hydrogen Bonds Around MobileRegions

[0419] Mobility of regions of a protein may be required for activity.The mobility of the α5,6 region, the putative channel-forming region ofCry3Bb, may be improved by decreasing the number of hydrogen bonds,including salt bridges (hydrogen bonds between oppositely charged aminoacid side chains), between helices 5-6 and any other part of themolecule or dimer structure. These hydrogen bonds may impede themovement of the two helices. Decreasing the number of hydrogen bonds andsalt bridges may improve biological activity. Replacement ofhydrogen-bonding amino acids with hydrophobic residues must be done withcaution to avoid creating continuous hydrophobic surfaces betweenhelices 5-6 and any other part of the dimer. This may decrease mobilityby increasing hydrophobic surface interactions.

[0420] 5.9.1 Cry3Bb.11222 And Cry3Bb.11223

[0421] Tyr230 is located on helix 6 and, in the quaternary dimerstructure of Cry3Bb, this amino acid is coordinated with Tyr230 from theadjacent molecule. Three hydrogen bonds are formed between the twohelices 6 in the two monomers because of this single amino acid. Inorder to improve the flexibility of helices 5-6, the helicestheoretically capable of penetrating the membrane and forming an ionchannel, the hydrogen bonds across the dimer were removed by changingthis amino acid and a corresponding increase in biological activity wasobserved. The designed Cry3Bb proteins, Cry3Bb.11222 and Cry3Bb.EG11223,show a 4-fold and 2.8-fold increase in SCRW activity, respectively,compared to WT.

[0422] 5.9.2 Cry3Bb.11051

[0423] Designed Cry3Bb protein Cry3Bb.11051 has amino acid change K189Gin lα4,5 of domain 1. In the WT Cry3Bb structure, the exposed side chainof K189 is close enough to the exposed side change of E123, located inlα2B,3, to form hydrogen bonds. Substitution of K189 with glycine, asfound in this position in Cry3A, removes the possibility of hydrogenbond formation at this site and results in a protein with a bioactivitythree-fold greater than WT Cry3Bb.

[0424] 5.9.3 Cry3Bb.11227, Cry3Bb.11241 and Cry3Bb.11242

[0425] Amino acid Q238, located in helix 6 of Cry3Bb, has beenidentified as a residue that, by its large size and hydrogen bonding toR290, blocks complete hydration of the space between helix 6 and helix4. Substitution of R290 with amino acids that do not form hydrogen bondsor that have side chains that can not span the physical distance tohydrogen bond with Q238 may increase the flexibility of thechannel-forming region. Designed proteins Cry3Bb.11227 (R290N),Cry3Bb.11241 (R290L) and Cry3Bb.11242 (R290V) show increased activitiesof approximately 2-fold, 2.6-fold and 2.5-fold, respectively, againstSCRW larvae compared to WT

5.10 Example 10

[0426] Design Method 4: Loop Analysis and Loop Design Around FlexibleHelices

[0427] Loop regions of a protein structure may be involved in numerousfunctions of the protein including, but not limited to, channelformation, quaternary structure formation and maintenance, and receptorbinding. Cry3Bb is a channel-forming protein. The availability of theion channel-forming helices of δ-endotoxins to move into the bilayerdepend upon the absence of forces that hinder the process. One of theforces possibly limiting this process is the steric hindrance of aminoacid side chains in loop regions around the critical helices. Theliterature suggests that in at least one other bacterial toxin, not a B.thuringiensis toxin, the toxin molecule opens up or, in scientificterms, loses some of the quaternary structure to expose amembrane-active region (Cramer et al., 1990). This literature does notteach how to improve the probability of this event occurring and it isnot known if B. thuringiensis toxins use this same process to penetratethe membrane. Reducing the steric hindrance of the amino acid sidechains in these critical regions by reducing size or altering side chainpositioning with the corresponding increase in biological activity wasthe inventive step.

[0428] 5.10.1 Analysis of the Loop Between Helices 3 and 4(Cry3Bb.11032)

[0429] The inventors have discovered that the first three helices ofdomain one could be cleaved from the rest of the toxin by proteolyticdigestion of the loop between helices α3 and α4 (Cry3Bb.60). Initialefforts to truncate the cry3Bb gene to produce this shortened, thoughmore active Cry3Bb molecule, failed. For unknown reasons, B.thuringiensis failed to synthesize this 60-kDa molecule. It was thenreasoned that perhaps the first three helices of domain 1 did not haveto be proteolytically removed, or equivalently, the protein did not haveto be synthesized in this truncated form to take advantage of theCry3Bb.60 design. It was observed that the protein Cry3A had a smallamino acid near the lα3,4 that might impart greater flexibility in theloop region thereby permitting the first three helices of domain 1 tomove out of the way, exposing the membrane-active region. By designing aCry3Bb molecule with a glycine residue near this loop, the sterichindrance of residues in the loop might be lessened. The redesignedprotein, Cry3Bb.11032, has the amino acid change D165G, which replacesthe larger aspartate residue (average mass of 115.09) with the smallestamino acid, glycine (average mass of 57.05). The activity ofCry3Bb.11032 is approximately 3-fold greater than that of the WTprotein. In this way, the loop between helices α3 and α4 was rationallyredesigned with a corresponding increase in the biological activity.

[0430] 5.10.2 Cry3Bb.11051

[0431] The loop region connecting helices α4 and α5 in Cry3Bb must beflexible so that the channel-forming helices α5-α6 can penetrate intothe membrane. It was noticed that Cry3A has a glycine residue in themiddle of this loop that may impart greater flexibility. Thecorresponding change, K189G, was made in Cry3Bb and the resulting,designed protein, Cry3Bb.11051, exhibits a 3-fold increase in activityagainst SCRW larvae compare to WT Cry3Bb.

[0432] 5.10.3 Analysis of the Loop Between β-Strand 1 and Helix 8(Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11232, Cry3Bb.11233,Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, and Cry3Bb.11239)

[0433] The loop region located between β strand 1 of domain 2 and αhelix 8 in domain 2 is very close to the loop between α helices 6 and 7in domain 1. Some of the amino acids side chains of lβ1,α8 appear asthough they may sterically impede movement of lα6,7. Since lα6,7 must beflexible for the channel-forming helices α5-α6 to insert into themembrane, it was thought that re-engineering this loop may change thepositioning of the side chains resulting in less steric hindrance. Thiswas accomplished creating proteins with increased biological activitiesranging from 2.2 to 5.4 times greater than WT. These designed toxinproteins and their amino acid changes are listed in Table 2 asCry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11232, Cry3Bb.11233,Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, and Cry3Bb.11239.

[0434] 5.10.4 Analysis of the Loop Between Helix 7 and β-Strand 1(Cry3Bb.11227, Cry3Bb.11234, Cry3Bb.11241, Cry3Bb.11242, andCry3Bb.11036)

[0435] If Cry3Bb is similar to a bacterial toxin which must open up toexpose a membrane active region for toxicity, it is possible that otherhelices in addition to the channel-forming helices must also changepositions. It was reasoned that, if helices α5-α6 insert into themembrane, than helix α7 may have to change positions also. It was shownin example 4.4.3 that increasing flexibility between helix α6 and α7 canincrease activity, greater flexibility in the loop following helix α7,lα7,β1 may also increase bioactivity. Alterations to the lα7,β1 regionof Cry3Bb resulted in the isolation of several proteins with increasedactivities ranging from 1.9 to 4.3 times greater than WT. These designedproteins are listed in Table 7 as Cry3Bb.11227, Cry3Bb.11234,Cry3Bb.11241, Cry3Bb.11242, and Cry3Bb.11036.

5.11 Example 11

[0436] Design Method 5: Loop Design Around β Strands and β Sheets

[0437] Loop regions of a protein structure may be involved in numerousfunctions of the protein including, but not limited to, channelformation, quaternary structure formation and maintenance, and receptorbinding. A binding surface is often defined by a number of loops, as isthe case with immunoglobulin G (IgG) (see Branden and Tooze, 1991, forreview). What can not be determined at this point, however, is whatloops will be important for receptor interactions just by looking at thestructure of the protein in question. Since a receptor has not beenidentified for Cry3Bb, it is not even possible to compare the structureof Cry3Bb with other proteins that have the same receptor for structuralsimilarities. To identify Cry3Bb loops that contribute to receptorinteractions, random mutagenesis was performed on surface-exposed loops.

[0438] As each loop was altered, the profile of the overallbioactivities of the resultant proteins were examined and compared. Theloops, especially in domain 2 which appears to be unnecessary forchannel activity, fall into two categories: (1) loops that could bealtered without much change in the level of bioactivity of the resultantproteins and (2) loops where alterations resulted in overall loss ofresultant protein bioactivity. Using this design method, it is possibleto identify several loops important for activity.

[0439] 5.11.1 Analysis of Loop β 2,3

[0440] Semi-random mutagenesis of the loop region between β strands 2and 3 resulted in the production of structurally stable toxin proteinswith significantly reduced activities against SCRW larvae. The lβ2,3region is highly sensitive to amino acid changes indicating thatspecific amino acids or amino acid sequences are necessary for toxinprotein activity. It is conceivable, therefore, that specific changes inthe lβ2,3 region will increase the binding and, therefore, the activityof the redesigned toxin protein.

[0441] 5.11.2 Analysis of Loop β 6,7

[0442] Semi-random mutations introduced to the loop region between βstrands 6 and 7 resulted in structurally stable proteins with an overallloss of SCRW bioactivity. The lβ6,7 region is highly sensitive to aminoacid changes indicating that specific amino acids or amino acidsequences are necessary for toxin protein activity. It is conceivable,therefore, that specific changes in the lβ6,7 region will increase thebinding and, therefore, the activity of the redesigned toxin protein.

[0443] 5.11.3 Analysis of Loop β 10,11

[0444] Random mutations to the loop region between β strands 10 and 11resulted in proteins having an overall loss of SCRW bioactivity. Loopβ10,11 is structurally close to and interacts with loops β2,3 and β6,7.Specific changes to individual residues within the lβ10,11 region mayalso result in increased interaction with the insect membrane,increasing the bioactivity of the toxin protein.

[0445] 5.11.4 Cry3Bb.11095

[0446] Loops β2,3, β6,7 and β10,11 have been identified as important forbioactivity of Cry3Bb. The 3 loops are surface-exposed and structurallyclose together. Amino acid Q348 in the WT structure, located in β-strand2 just prior to lβ2,3, does not form any intramolecular contacts.However, replacing Q348 with arginine (Q348R) results in the formationof 2 new hydrogen-bonds between R348 and the backbone carbonyls of R487and R488, both located in lβ10,11. The new hydrogen bonds may act tostabilize the structure formed by the 3 loops. The designed proteincarrying this change, Cry3Bb.11095, is 4.6-fold more active than WTCry3Bb.

5.12 Example 12

[0447] Design Method 6: Identification and Re-Design of ComplexElectrostatic Surfaces

[0448] Interactions of proteins include hydrophobic interactions (e.g.,Van der Waals forces), hydrophilic interactions, including those betweenopposing charges on amino acid side chains (salt bridges), and hydrogenbonding. Very little is known about δ-endotoxin and receptorinteractions. Currently, there are no literature reports identifying thetypes of interactions that predominate between B. thuringiensis toxinsand receptors.

[0449] Experimentally, however, it is important to increase the strengthof the B. thuringiensis toxin-receptor interaction and not permit theprecise determination of the chemical interaction to stand in the way ofimproving it. To accomplish this, the electrostatic surface of Cry3Bbwas defined by solving the Poisson-Boltzman distribution around themolecule. Once this electrically defined surface was solved, it couldthen be inspected for regions of greatest diversity. It was reasonedthat these electrostatically diverse regions would have the greatestprobability of participating in the specific interactions between the B.thuringiensis toxin proteins and the receptor, rather than more generaland non-specific interactions. Therefore, these regions were chosen forredesign, continuing to increase the electrostatic diversity of theregions. In addition, examination of the electrostatic interactionaround the putative channel forming region of the toxin created insightsfor redesign. This includes identification of an electropositive residuein an otherwise negatively charged conduit (see example 4.6.1).

[0450] 5.12.1 R290 (Cry3Bb.11227, Cry3Bb.11241, and Cry3Bb.11242)

[0451] Examination of the Cry3Bb dimer interface along the domain 1 axissuggested that a pore or conduit for cations might be formed between themonomers. Electrostatic examination of this axis lent additionalcredibility to this suggestion. In fact, the hypothetical conduit isprimarily negatively charged, an observation consistent with thebiophysical analysis of cation-selective, δ-endotoxin channels. If acation channel were formed along the axis of the dimer, then the cationcould move between the monomers relatively easily with only onesignificant hurdle. A positively charged arginine residue (R290) lies inthe otherwise negatively charged conduit. This residue could impede thecation movement through the channel. Based on this analysis, R290 waschanged to uncharged residues. The bioactivity of redesigned proteinsCry3Bb.11227 (R290N), Cry3Bb.11241 (R290L) and Cry3Bb.11242 (R290V) wasimproved approximately 2-fold, 2.6-fold and 2.5-fold, respectively.

[0452] 5.12.2 Cry3Bb.60

[0453] Trypsin digestion of solubilized Cry3Bb yields a stable,truncated protein with a molecular weight of 60 kDa (Cry3Bb.60). Trypsindigestion occurs on the carboxyl side of residue R159, effectivelyremoving helices 1 through 3 from the native Cry3Bb structure. Thecleavage of the first 3 helices exposes an electrostatic surfacedifferent than those found in the native structure. The new surface hasa combination of hydrophobic, polar and charged characteristics that mayplay a role in membrane interactions. The bioactivity of Cry3Bb.60 is3.6-fold greater than that of WT Cry3Bb.

5.13 Example 13

[0454] Design Method 7: Identification and Removal of Metal BindingSites

[0455] The literature teaches that the in vitro behavior of B.thuringiensis toxins can be increased by chelating divalent cations fromthe experimental system (Crawford and Harvey 1988). It was not known,however, how these divalent cations inhibited the in vitro activity.Crawford and Harvey (1988) demonstrated that the short circuit currentacross the midgut was more severely inhibited by B. thuringiensis in thepresence of EDTA, a chelator of divalent ions, than in the absence ofthis agent, thus suggesting that this step in the mode of action of B.thuringiensis could be potentiated by removing divalent ions. Similarobservations were made using black-lipid membranes and measuring anincrease in the current created by the δ-endotoxins in the presence ofEDTA to chelate divalent ions. There were at least three possibleexplanations for these observations. The first explanation could be thatthe divalent ions are too large to move through a ion channel moresuitable for monovalent ions, thereby blocking the channel. Second, thedivalent ions may cover the protein in the very general way, therebybuffering the charge interactions required for toxin membraneinteraction and limiting ion channel activity. The third possibility isthat a specific metal binding site exists on the protein and, whenoccupied by divalent ions, the performance of the ion channel isimpaired. Although the literature could not differentiate the value ofone possibility over another, the third possibility led to an analysisof the Cry3Bb structure searching for a specific metal binding site thatmight alter the probability that a toxin could form an ion channel.

[0456] 5.13.1 H231 (Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225, andCry3Bb.11226)

[0457] A putative metal binding site is formed in the Cry3Bb dimerstructure by the H231 residues of each monomer. The H231 residues,located in helix α6, lie adjacent to each other and close to the axis ofsymmetry of the dimer. Removal of this site by replacement of histidinewith other amino acids was evaluated by the absence of EDTA-dependention channel activity. The bioactivities of the designed toxin proteins,Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225 and Cry3Bb.11226, are increased4-, 5-, 3.6- and 3-fold, respectively, over that of WT Cry3Bb. Theirrespective amino acid changes are listed in Table 2.

5.14 Example 14

[0458] Design Method 8: Alteration of Quaternary Structure

[0459] Cry3Bb can exist in solution as a dimer similar to a relatedprotein, Cry3A (Walters et al., 1992). However, the importance of thedimer to biological activity is not known because the toxin as a monomeror as a higher order structure has not been seriously evaluated. It isassumed that specific amino acid residues contribute to the formationand stability of the quaternary structure. Once a contributing residueis identified, alterations can be made to diminish or enhance the effectof that residue thereby affecting the interaction between monomers.Channel activity is a useful way, but by no means the only way, toassess quaternary structure of Cry3Bb and its derivatives. It has beenobserved that Cry3Bb creates gated conductances in membranes that growin size with time, ultimately resulting in large pores in the membrane(the channel activity of WT Cry3Bb is described in Section 12.1). Italso has been observed that Cry3A forms a more stable dimer than Cry3Bband coincidentally forms higher level conductances faster (FIG. 10).This observation led the inventors to propose that oligomerization andion channel formation (conductance size and speed of channel formation)were related. Based on this observation Cry3Bb was re-engineered to makelarger and more stable oligomers at a faster rate. It is assumed in thisanalysis that the rate of ion channel formation and growth mirrors thisprocess. It is also possible that changes in quaternary structure maynot affect channel activity alone or at all. Alterations to quaternarystructure may also affect receptor interactions, protein processing inthe insect gut environment, as well as other aspects of bioactivityunknown.

[0460] 5.14.1 Cry3Bb.11048

[0461] Comparative structural analysis of Cry3A and Cry3Bb led to theidentification of structural differences between the two toxins in theion channel-forming domain; specifically, an insertion of one amino acidbetween helix 2a and helix 2b in Cry3Bb. Removal of this additionalamino acid in Cry 3B2, A104, and a D103E substitution, as in Cry3A,resulted in loss of channel gating and the formation of symmetricalpores. Once the pores are formed they remain open and allow a steadyconductance ranging from 25-130 pS. This designed protein, Cry3Bb.11048,is 4.3 times more active than WT Cry3Bb against SCRW larvae.

[0462] 5.14.2 Oligomerization of Cry3Bb.60

[0463] Individual molecules of Cry3Bb or Cry3Bb.60 can form a complexwith another like molecule. Oligomerization of Cry3Bb is demonstrated bySDS-PAGE, where samples are not heated in sample buffer prior to loadingon the gel. The lack of heat treatment allows some nondenatured toxin toremain. Oligomerization is visualized following Coomassie staining bythe appearance of a band at 2 times the molecular weight of the monomer.The intensity of the higher molecular weight band reflects the degree ofoligomerization. The ability of Cry3Bb to form an oligomer is notreproducibly apparent. The complex cannot be repeatedly observed toform. Cry3Bb.60, however, forms a significantly greater amount of ahigher molecular weight complex (120 kDa). These data suggest thatCry3Bb.60 more readily forms the higher order complex than Cry3Bb alone.Cry3Bb.60 also forms ion channels with greater frequency than WT Cry3Bb(see Section 5.12.9).

[0464] 5.14.3 Cry3Bb.11035

[0465] Changes were made in Cry3Bb to reflect the amino acid sequence inCry3A at the end of lα3,4 and in the beginning of helix 4. These changesresulted in the designed protein, Cry3Bb.11035, that, unlike wild typeCry3Bb, forms spontaneous channels with large conductances. Cry3Bb.11035is also approximately three times more active against SCRW larvae thanWT Cry3Bb. Cry3Bb.11035 and its amino acid changes are listed in Table10.

[0466] 5.14.4 Cry3Bb.11032

[0467] Cry3Bb.11032 was altered at residue 165 in helix α4, changing anasparate to glycine, as found in Cry3A. Cry3Bb.11032 is three-fold moreactive than WT Cry3Bb. The channel activity of Cry3Bb.11032 is much likeCry3Bb except when the designed protein is artificially incorporatedinto the membrane. A 16-fold increase in the initial channelconductances is observed compared to WT Cry3Bb (see Section 5.12.2).This increase in initial conductance presumably is due to enhancedquaternary structure, stability or higher-order structure.

[0468] 5.14.5 EG11224

[0469] In the WT Cry3Bb dimer structure, histidine, at position 231 indomain 1, makes hydrogen bond contacts with D288 (domain 1), Y230(domain 1), and, through a network of water molecules, also makescontacts to D610 (domain 3), all of the opposite monomer. D610 and K235(domain 1) also make contact. Replacing the histidine with an arginine,H231R, results, in one orientation, in the formation of a salt bridge toD610 of the neighboring monomer. In a second orientation, the contactswith D288 of the neighboring monomer, as appear in the WT structure, areretained. In either orientation, R231 does not hydrogen bond to Y230 ofthe opposite monomer but does make contact with K235 which retains iscontacts to K610 (V. Cody, research communication). The shiftinghydrogen bonds have changed the interactions between the differentdomains of the protein in the quaternary structure. Overall, fewerhydrogen bonds exist between domains 1 of the neighboring monomers and amuch stronger bond has been formed between domains 1 and 3. Channelactivity was found to be altered. Cry3Bb.11224 produces small, quicklygating channels like Cry3Bb. However, unlike WT Cry3Bb, Cry3Bb.11224does not exhibit β-mercaptoethanol-dependent activation. Replacing H231with arginine resulted in a designed Cry3Bb protein, Cry3Bb.11224,exhibiting a 5-fold increase in bioactivity.

[0470] 5.14.6 Cry3Bb.11226

[0471] Cry3Bb.11226 is similar to Cry3Bb.11224, discussed in Section4.8.5, in that the histidine at position 23 1 has been replaced. Theamino acid change, H231T, results in the loss of β-mercaptoethanoldependent activation seen with WT Cry3Bb (see Section 5.12.1). Thereplacement of H231, a putative metal binding site, changes theinteraction of regions in the quaternary structure resulting in adifferent type of channel activity. Cry3Bb.11226 is three-fold moreactive than WT Cry3Bb.

[0472] 5.14.7 Cry3Bb.11221

[0473] Cry3Bb.11221 has been re-designed in the lα3,4 region of Cry3Bb.The channels formed by Cry3Bb.11221 are much more well resolved than theconductances formed by WT Cry3Bb (see Section 5.12.6). Cry3Bb.11221exhibits a 6.4-fold increase in bioactivity over that of WT Cry3Bb. Theamino acid changes found in Cry3Bb.11221 are listed in Table 2.

[0474] 5.14.8 Cry3Bb.11242

[0475] The designed protein, Cry3Bb.11242, carrying the alterationR290V, forms small conductances immediately which grow rapidly andsteadily to large conductances in about 3 min (see Section 5.12.7). Thisis contrast to WT Cry3Bb channels which take 30-45 min to appear andgrow slowly over hours to large conductances. Cry3Bb.11242 also exhibitsa 2.5-fold increase in bioactivity compared to WT Cry3Bb.

[0476] 5.14.9 Cry3Bb.11230

[0477] Cry3Bb.11230, unlike WT Cry3Bb, forms well resolved channels withlong open states. These channels reach a maximum conductance of 3000 pSbut do not continue to grow with time. Cry3Bb.11230 has been re-designedin the lβ1,α8 region of Cry3Bb and exhibits almost a 5-fold increase inactivity against SCRW larvae (Table 9) and a 5.4-fold increase againstWCRW larvae (Table 10) compared to WT Cry3Bb. The amino acid changesfound in Cry3Bb.11230 are listed in Table 2.

5.15 Example 15

[0478] Design Method 9: Design of Structural Residues

[0479] The specific three-dimensional structure of a protein is held inplace by amino acids that may be buried or otherwise removed from thesurface of the protein. These structural determinants can be identifiedby inspection of forces responsible for the surface structurepositioning. The impact of these structural residues can then beenhanced to restrict molecular motion or diminished to enhance molecularflexibility.

[0480] 5.15.1 Cry3Bb.11095

[0481] Loops β2,3, β6,7 and β10,11, located in domain 2 of Cry3Bb, havebeen identified as important for bioactivity. The three loops aresurface-exposed and structurally close together. Amino acid Q348 in theWT structure, located in β-strand 2 just prior to lβ2,3, does not formany intramolecular contacts. However, replacing Q348 with arginine(Q348R) results in the formation of 2 new hydrogen-bonds between R348and the backbone carbonyls of R487 and R488, both located in lβ10,11.The new hydrogen bonds may act to stabilize the structure formed by thethree loops. Certainly, the structure around R348 is more tightly packedas determined by X-ray crystallography. The designed protein carryingthis change, Cry3Bb.11095, is 4.6-fold more active than WT Cry3Bb.

5.16 Example 16

[0482] Design Method 10: Combinatorial Analysis and Mtuagenesis

[0483] Individual sites in the engineered Cry3Bb molecule can be usedtogether to create a Cry3Bb molecule with activity even greater than theactivity of any one site. This method has not been precisely applied toany δ-endotoxin. It is also not obvious that improvements in two sitescan be pulled together to improve the biological activity of theprotein. In fact, data demonstrates that improvements to 2 sites, whenpulled together into a single construct, do not necessarily furtherimprove the biological activity of Cry3Bb. In some cases, thecombination resulted in decreased protein stability and/or activity.Examples of proteins with site combinations that resulted in improvedactivity compared to WT Cry3Bb but decreased activity compared to 1 ormore of the “parental” proteins are Cry3Bb.11235, 11046, 11057 and11058. Cry3Bb.11082, which contains designed regions from 4 parentalproteins, retains the level of activity from the most active parentalstrain (Cry3Bb.11230) but does not show an increase in activity. Theseproteins are listed in Table 7. The following are examples of instanceswhere combined mutations have significantly improved biologicalactivity.

[0484] 5.16.1 Cry3Bb.11231

[0485] Designed protein Cry3Bb.11231 contains the alterations found inCry3Bb.11224 (H231R) and Cry3Bb.11228 (changes in lβ1,α8). Thecombination of amino acid changes found in Cry3Bb.11231 results in anincrease in bioactivity against SCRW larvae of approximately 8-fold overthat of WT Cry3Bb (Table 2). This increase is greater than exhibited byeither Cry3Bb.11224 (5.0×) or Cry3Bb.11228 (4.1×) alone. Cry3Bb.11231was also exhibits an 12.9-fold increase in activity compared to WTCry3Bb against WCRW larvae (Table 10).

[0486] 5.16.2 Cry3Bb.11081

[0487] Designed Cry3Bb protein Cry3Bb.11081 was constructed by combiningthe changes found in Cry3Bb.11032 and Cry3Bb.11229 (with the exceptionof Y318C). Cry3Bb.11081 a 6.1-fold increase in activity over WT Cry3Bb;a greater increase in activity than either of the individual parentalproteins, Cry3Bb.11032 (3.1-fold) and Cry3Bb.11229 (2.5-fold).

[0488] 5.16.3 Cry3Bb.11083

[0489] Designed Cry3Bb protein Cry3Bb.11083 was constructed by combiningthe changes found in Cry3Bb.11036 and Cry3Bb.11095. Cry3Bb.11083exhibits a 7.4-fold increase in activity against SCRW larvae compared toWT Cry3Bb; a greater increase than either Cry3Bb.11036 (4.3×) orCry3Bb.11095 (4.6×). Cry3Bb.11083 also exhibits a 5.4-fold increase inactivity against WCRW larvae compared to WT Cry3Bb (Table 10).

[0490] 5.16.4 Cry3Bb.11084

[0491] Designed Cry3Bb protein Cry3Bb.11084 was constructed by combiningthe changes found in Cry3Bb.11032 and the S311L change found inCry3Bb.11228. Cry3Bb.11084 exhibits a 7.2-fold increase in activity overthat of WT Cry3Bb; a greater than either Cry3Bb.11032 (3.1×) orCry3Bb.11228 (4.1×).

[0492] 5.16.5 Cry3Bb.11098

[0493] Designed Cry3Bb protein Cry3Bb.11098 was constructed to containthe following amino acid changes: D165G, H231R, S311L, N313T, and E317K.The nucleic acid sequence is given in SEQ ID NO:107, and the encodedamino acid sequence is given in SEQ ID NO:108.

5.17 Example 17

[0494] Design Strategy 11: Alteration of Binding to Glycoproteins and toWCRW Brush Border Membranes

[0495] While the identity of receptor(s) for Cry3Bb is unknown, it isnonetheless important to increase the interaction of the toxin with itsreceptor. One way to improve the toxin-receptor interaction with knowingthe identity of the receptor is to reduce or eliminate non-productivebinding to other biomolecules. The inventors have observed that Cry3Bbbinds non-specifically to bovine serum albumin (BSA) that has beenglycosylated with a variety of sugar groups, but not to non-glycosylatedBSA. Cry3A, which is not active on Diabrotica species, shows similar buteven greater binding to glycosylated-BSA. Similarly, Cry3A shows greaterbinding to immobolized WCRW brush border membrane (BBM) than does WTCry3Bb, suggesting that much of the observed binding is non-productive.It was reasoned that the non-specific binding to WCRW BBM occurs viaglycosylated proteins, and that binding to both glycosylated-BSA andWCRW BBM is non-productive in reaction pathway to toxicity. Thereforereduction or elimination of that binding would lead to enhanced bindingto the productive receptor and to enhanced toxicity. Potential bindingsites for sugar groups were targeted for redesign to reduce thenon-specific binding of Cry3Bb to glycoproteins and to immobilized WCRWBBM.

[0496] 5.17.1 Cry3Bb.60

[0497] Cry3Bb-60, in which Cry3Bb has been cleaved at R159 in lα3,4,shows decreased binding to glycosylated-BSA and decreased binding toimmobilized WCRW BBM. Cry3Bb-60 shows a 3.6-fold increase in bioactivityrelative to WT Cry3Bb.

[0498] 5.17.2 Alterations to lα3,4 (Cry3Bb.11221)

[0499] Cry3Bb.11221 has been redesigned in the lα3,4 region of domain 1,which is the region in which Cry3Bb is cleaved to produce Cry3Bb-60.Cry3Bb.11221 also shows decreased binding to both glycosylated-BSA andimmobilized WCRW BBM, and exhibits a 6.4-fold increase in bioactivityover that of WT Cry3Bb. Together with data for Cry3Bb.60 (section5.17.1) these data suggest that this loop region contributessubstantially to non-productive binding of the toxin.

[0500] 5.17.3 Alteration of lβ1,α8 (Cry3Bb.11228,11230,11237 and 11231)

[0501] The lβ1,α8 region of Cry3Bb has been re-engineered to increasehydration (section 4.2.4) and enhance flexibility (section 4.4.3).Several proteins altered in this region, Cry3Bb.11228,11230, and 11237demonstrate substantially lower levels of binding both glycosylated-BSAand immobilized WCRW BBM, and also show between 4.1- and 4.5-foldincreases in bioactivity relative to WT Cry3Bb.

[0502] 5.17.4 Binding Activity

[0503] The tendencies of Cry3Bb and some of its derivatives to bind toglycosylated-BSA and to WCRW BBM were determined using a BIAcore™surface plasmon resonance biosensor. For glycosylated-BSA binding, theglycosylated protein was immobilized using standard NHS chemistry to aCM5 chip (BIAcore), and the solubilized toxin was injected over theglycosylated-BSA surface. To measure binding to WCRW BBM, brush bordermembrane vesicles (BBMV) purified from WCRW midguts (English et al.,1991) were immobilized on an HPA chip (BIAcore) then washed with either10 mM KOH or with 40 mM β-octylglucoside. The solubilized toxin was theninjected over the resulting hybrid bilayer surface to detect binding.Protein concentration were determined by Protein Dye Reagent assay(BioRad) or BCA Protein Assay (Pierce). Other methods may also be usedto determine the same binding information. These include, but are notlimited to, ligand blot experiments using labeled toxin, labeledglycosylated protein, or anti-toxin antibodies, affinity chromatography,and in vitro binding of toxin to intact BBMV.

5.18 Example 18

[0504] Construction of Plasmids with WT Cry3Bb Sequences

[0505] Standard recombinant DNA procedures were performed essentially asdescribed by Sambrook et al., (1989).

[0506] 5.18.1 pEG1701

[0507] pEG1701 (FIG. 11), contained in EG11204 and EG11037, wasconstructed by inserting the SphI-PstI fragment containing the cry3Bbgene and the cry1F terminator from pEG911 (Baum, 1994) into theSphI-PstI site of pEG854.9 (Baum et al., 1996), a high copy number B.thuringiensis-E. coli shuttle vector.

[0508] 5.18.2 pEG1028

[0509] pEG1028 contains the HindIII fragment of cry3Bb from pEG1701cloned into the multiple cloning site of pTZ18U at HindIII.

5.19 Example 19

[0510] Construction of Plasmids with Altered Cry3Bb Genes

[0511] Plasmid DNA from E. coli was prepared by the alkaline lysismethod (Maniatis et al., 1982) or by commercial plasmid preparation kits(examples: PERFECTprep™ kit, 5 Prime-3 Prime, Inc., Boulder Colo.;QIAGEN plasmid prep kit, QIAGEN Inc.). B thuringiensis plasmids wereprepared from cultures grown in brain heart infusion plus 0.5% glycerol(BHIG) to mid logarithmic phase by the alkaline lysis method. Whennecessary for purification, DNA fragments were excised from an agarosegel following electrophoresis and recovered by glass milk using aGeneclean II® kit (BIO 101 Inc., La Jolla, Calif.). Alteration of thecry3Bb gene was accomplished using several techniques includingsite-directed mutagenesis, triplex PCR™, quasi-random PCR™ mutagenesis,DNA shuffling and standard recombinant techniques. These techniques aredescribed in Sections 6.1, 6.2, 6.3, 6.4 and 6.5, respectively. The DNAsequences of primers used are listed in Section 7.

5.20 Example 20

[0512] Site-Directed Mutagenesis

[0513] Site-directed mutagenesis was conducted by the protocolsestablished by Kunkle (1985) and Kunkle et al. (1987) using theMuta-Gene™ M13 in vitro mutagenesis kit (Bio-Rad, Richmond, Calif.).Combinations of alterations to cry3Bb were accomplished by using theMuta-Gene™ kit and multiple mutagenic oligonucleotide primers.

[0514] 5.20.1 pEG1041

[0515] pEG1041, contained in EG11032, was constructed using theMuta-Gene™ kit, primer C, and single-stranded pEG1028 as the DNAtemplate. The resulting altered cry3Bb DNA sequence was excised as aPflMI DNA fragment and used to replace the corresponding DNA fragment inpEG1701.

[0516] 5.20.2 pEG1046

[0517] pEG1046, contained in EG11035, was constructed using theMuta-Gene™ kit, primer D, and single-stranded pEG1028 as the DNAtemplate. The resulting altered cry3Bb DNA sequence was excised as aPflMI DNA fragment and used to replace the corresponding DNA fragment inpEG1701.

[0518] 5.20.3 pEG1047

[0519] pEG1047, contained in EG11036, was constructed using theMuta-Gene™ kit, primer E, and single-stranded pEG1028 as the DNAtemplate. The resulting altered cry3Bb DNA sequence was excised as aPflMI DNA fragment and used to replace the corresponding DNA fragment inpEG1701.

[0520] 5.20.4 pEG1052

[0521] pEG1052, contained in EG11046, was constructed using theMuta-Gene™ kit, primers D and E, and single-stranded pEG1028 as the DNAtemplate. The resulting altered cry3Bb DNA sequence was excised as aPflMI DNA fragment and used to replace the corresponding DNA fragment inpEG1701.

[0522] 5.20.5 pEG1054

[0523] pEG1054, contained in EG11048, was constructed using theMuta-Gene™ kit, primer F, and single-stranded pEG1028 as the DNAtemplate. The resulting altered cry3Bb DNA sequence was excised as aPflMI DNA fragment and used to replace the corresponding DNA fragment inpEG1701.

[0524] 5.20.6 pEG1057

[0525] pEG1057, contained in EG11051, was constructed using theMuta-Gene™ kit, primer G, and single-stranded pEG1028 as the DNAtemplate. The resulting altered cry3Bb DNA sequence was excised as aPflMI DNA fragment and used to replace the corresponding DNA fragment inpEG1701.

5.21 Example 21

[0526] Triplex PCR™

[0527] Triplex PCR™ is described by Michael (1994). This method makesuse of a thermostable ligase to incorporate a phosphorylated mutagenicprimer into an amplified DNA fragment during PCR™. PCR™ was performed ona Perkin Elmer Cetus DNA Thermal Cycler (Perkin-Elmer, Norwalk, Conn.)using a AmpliTaq™ DNA polymerase kit (Perkin-Elmer) and SphI-linearizedpEG1701 as the template DNA. PCR™ products were cleaned using commercialkits such as Wizard™ PCR™ Preps (Promega, Madison, Wis.) and QIAquickPCR™ Purification kit (QIAGEN Inc., Chatsworth, Calif.).

[0528] 5.21.1 pEG1708 and pEG1709

[0529] pEG1708 and pEG1709, contained in EG11222 and EG11223,respectively, were constructed by replacing the PflMI-PflAI fragment ofcry3Bb in pEG1701 with PflMI-digested and gel purified PCR™ fragmentaltered at cry3Bb nucleotide positions 688-690. encoding amino acidY230. Random mutations were introduced into the Y230 codon by triplexPCR™. Mutagenic primer MVT095 was phosphorylated and used together withoutside primer pair FW001 and FW006. Primer MVT095 also contains asilent mutation at position 687, changing T to C, which, uponincorporation, introduces an additional EcoRI site into pEG1701.

[0530] 5.21.2 pEG1710 pEG1711 and pEG1712

[0531] Plasmids pEG1710, pEG1711 and pEG1712, contained in EG11224,EG11225 and EG11226, respectively, were created by replacing thePflMI-PflMI fragment of the cry3Bb gene in pEG1701 with PflMI-digestedand gel purified PCR™ fragment altered at cry3Bb nucleotide positions690-692, encoding H231. Random mutations were introduced into the H231codon by triplex PCR™. Mutagenic primer MVT097 was phosphorylated andused together with outside primer pair FW001 and FW006. Primer MVT097also contains a T to C sequence change at position 687 which, uponincorporation, results in an additional EcoRI site by silent mutation.

[0532] 5.21.3 pEG1713 and pEG1727

[0533] pEG1713 and pEG1727, contained in EG11227 and EG11242,respectively, were constructed by replacing the PflMI-PflMi fragment ofthe cry3Bb gene in pEG1701 with PflMI-digested and gel purified PCR™fragment altered at cry3Bb nucleotide positions 868-870, encoding aminoacid R290. Triplex PCR™ was used to introduce random changes into theR290 codon. The mutagenic primer, MVT091, was designed so that thenucleotide substitutions would result in approximately 36% of thesequences encoding amino acids D or E. MVT091 was phosphorylated andused together with outside primer pair FW001 and FW006.

[0534] 5.22 Example 22

[0535] Quasi-Random PCR™ Mutagenesis

[0536] Quasi-random mutagenesis combines the mutagenic PCR™ techniquesdescribed by Vallette et al. (1989), Tomic et al. (1990) and LaBean andKauffman (1993). Mutagenic primers, sometimes over 70 nucleotides inlength, were designed to introduce changes over nucleotide positionsencoding for an entire structural region, such as a loop. Degeneratecodons typically consisted of a ratio of 82% WT nucleotide plus 6% eachof the other 3 nucleotides per position to semi-randomly introducechanges over the target region (LaBean and Kauffman, 1993). Whenpossible, natural restriction sites were utilized; class 2s enzymes wereused when natural sites were not convenient (Stemmer and Morris, 1992,list additional restriction enzymes useful to this technique). PCR™ wasperformed on a Perkin Elmer Cetus DNA Thermal Cycler (Perkin-Elmer,Norwalk, Conn.) using a AmpliTaq™ DNA polymerase kit (Perkin-Elmer) andSphI-linearized pEG1701 as the template DNA. Quasi-random PCR™amplification was performed using the following conditions: denaturationat 94° C. for 1.5 min.; annealing at 50° C. for 2 min. and extension at72° C. for 3 min., for 30 cycles. The final 14 extension cycles wereextended an additional 25 s per cycle. Primers concentration was 20 μMper reaction or 40 μM for long, mutagenic primers. PCR™ products werecleaned using commercial kits such as Wizard™ PCR™ Preps (Promega,Madison. Wis.) and QIAquick PCR™ Purification kit (QIAGEN Inc.,Chatsworth, Calif.). In some instances PCR™ products were treated withKlenow Fragment (Promega) following the manufacturer's instructions tofill in any single base overhangs prior to restriction digestion.

[0537] 5.22.1 pEG1707

[0538] EG1707, contained in EG11221, was constructed by replacing thePflMI-PflMI fragment of the cry3Bb gene in pEG1701 with PflMI-digestedand gel purified PCR™ fragment altered at cry3Bb nucleotide positions460-480, encoding la3,4 amino acids 154-160. Primer MVT075, whichincludes a recognition site for the class 2s restriction enzyme BsaI,and primer FW006 were used to introduce changes into this region byquasi-random mutagenesis. Primers MVT076, also containing a BsaI site,and primer FW001 were used to PCR™ amplify a “linker” fragment.Following PCR™ amplification, both products were cleaned, end-filled,digested with BsaI and ligated to each other. Ligated fragment was gelpurified and used as template for PCR™ amplification using primer pairFW001 and FW006. PCR™ product was cleaned, digested with PflMI, gelpurified and ligated into PFTMI-digested and purified pEG1701 vectorDNA.

[0539] 5.22.2 pEG1720 and pEG1726

[0540] pEG1720 and pEG1726, contained in EG11234 and EG11241,respectively, were constructed by replacing the PflMI-PflMI fragment ofthe cry3Bb gene in pEG1701 with PflMI-digested and gel purified PCR™fragment altered at cry3Bb nucleotide positions 859-885, encoding lα7,β1amino acids 287-295. Quasi-random PCR™ mutagenesis was used to introducechanges into this region. Mutagenic primer MVT111, designed with a BsaIsite, and primer FW006 were used to introduce the changes. Primer pairMVT094, also containing a BsaI site, and FW001 were used to amplify thelinker fragment. The PCR™ products were digested with BsaI, gel purifiedthen ligated to each other. Ligated product was PCR™ amplified usingprimer pair FW001 and FW006, digested with PflMI.

[0541] 5.22.3 pEG1714, pEG1715, pEG1716, pEG1718, pEG1719, pEG1722,pEG1723, pEG1724 and pEG1725

[0542] pEG1714, pEG1715, pEG1716, pEG1718, pEG1719, pEG1722, pEG1723,pEG1724 and pEG1725, contained in EG11228, EG11229, EG11230, EG11232,EG11233, EG11236, EG11237, EG11238 and EG11239, respectively, wereconstructed by replacing the PflMI-PflMI fragment of the cry3Bb gene inpEG1701 with PflMI-digested and gel purified PCR™ fragment altered atcry3Bb nucleotide positions 931-954, encoding lβ1,α8 amino acids311-318. Quasi-random PCR™ mutagenesis was used to introduce changesinto this region using mutagenic primer MVT103 and primer FW006. PrimersFW001 and FW006 were used to amplify a linker fragment. The PCR™products were end-filled using Klenow and digested with BamHI. Thelarger fragment from the FW001-FW006 digest was gel purified thenligated to the digested MVT103-FW006 fragment. Ligated product was gelpurified and amplified by PCR™ using primer pair FW001 and FW006. Theamplified product was digested with PflMI and gel purified prior toligation into PflMI-digested and purified pEG1701 vector DNA.

[0543] 5.22.4 pEG1701.Lβ2.3

[0544] Plasmids carrying alterations of cry3Bb WT sequence atnucleotides 1051-1065, encoding structural region lβ2,3 of Cry3Bb, wereconstructed by replacing the MfuI-SpeI fragment of pEG1701 with isolatedMfuI- and SpeI-digested PCR™ product. The PCR™ product was generated byquasi-random PCR™ mutagenesis were mutagenic primer MVT081 was pairedwith FW006. These plasmids as a group are designated pEG1701.1β2,3.

[0545] 5.22.5 pEG1701.Lβ6,7

[0546] Plasmids containing mutations of the cry3Bb WT sequence atnucleotides 1234-1248, encoding structural region lβ6,7 of Cry3Bb, wereconstructed by replacing the MfuI-SpeI fragment of pEG1701 with isolatedMfuI- and SpeI-digested PCR™ product. The PCR™ product was generated byquasi-random PCR™ mutagenesis where mutagenic primer MVT085 was pairedwith primer WD115. Primer pair MVT089 and WD112 were used to amplify alinker fragment. Both PCR™ products were digested with TaqI and ligatedto each other. The ligation product was gel purified and PCR™ amplifiedusing primer pair MVT089 and FW006. The amplified product was digestedwith MluI and SpeI and ligated into MluI and SpeI digested and purifiedpEG1701 vector DNA. These plasmids as a group are designatedpEG1701.lβ6,7.

[0547] 5.22.6 pEG1701.Lβ10,11

[0548] Plasmids containing mutated cry3Bb sequences at nucleotides1450-1467, encoding structural region lβ10,11 of Cry3Bb, wereconstructed by replacing the SpeI-PstI fragment of pEG1701 with isolatedSpeI- and PstI-digested PCR™ product. The PCR™ product was generated byquasi-random PCR™ mutagenesis where mutagenic primer MVT105 was pairedwith primer MVT070. Primer pair MVT092 and MVT083 were used to generatea linker fragment. (MVT083 is a mutagenic oligo designed for anotherregion. The sequence changes introduced by MVT083 are removed followingrestriction digestion and do not impact the alteration of cry3Bb in thelβ10,11 region.) Both PCR™ products were digested with BsaI, ligatedtogether, and the ligation product PCR™ amplified with primer pairMVT083 and MVT070. The resulting PCR™ product was digested with SpeI andPstI, and gel purified. These plasmids as a group are designatedpEG1701.lβ10,11.

5.23 Example 23

[0549] DNA Shuffling

[0550] DNA-shuffling, as described by Stemmer (1994), was used tocombine individual alterations in the cry3Bb gene.

[0551] 5.23.1 pEG1084, pEG1085, pEG1086 and pEG1087

[0552] pEG1084, pEG1085, pEG1086, and pEG1087, contained in EG11081,EG11082, EG11083, and EG11084, respectively, were recovered fromDNA-shuffling. Briefly, PflMI DNA fragments were generated using primerset A and B and each of the plasmids pEG1707, pEG1714, pEG1715, pEG1716,pEG1041, pEG1046, pEG1047, and pEG1054 as DNA templates. The resultingDNA fragments were pooled in equal-molar amounts and digested withDNaseI and 50-100 bp DNA fragments were recovered from an agarose gel bythree successive freeze-thaw cycles: three min in a dry-ice ethanol bathfollowed by complete thawing at 50° C. The recovered DNA fragments wereassembled by primerless-PCR™ and PCR™-amplified using the primer set Aand B as described by Stemmer (1994). The final PCR™-amplified DNAfragments were cut with PflMI and used to replace the correspondingcry3Bb PflMI DNA fragment in pEG1701.

5.24 Example 24

[0553] Recombinant DNA Techniques

[0554] Standard recombinant DNA procedures were performed essentially asdescribed by Sambrook et al. (1989).

[0555] 5.24.1 pEG1717

[0556] pEG1717, contained in EG11231, was constructed by replacing thesmall BglII fragment of pEG1710 with the small BglII fragment frompEG1714.

[0557] 5.24.2 pEG1721

[0558] pEG1721, contained in EG11235, was constructed by replacing thesmall BglII fragment from pEG1710 with the small BglII fragment frompEG1087.

[0559] 5.24.3 pEG1063

[0560] pEG1062, contained in EG11057, was constructed by replacing theNcoI DNA fragment containing ori 43 from pEG1054 with the isolated NcoIDNA fragment containing ori 43 and the alterations in cry3Bb frompEG1046.

[0561] 5.24.4 pEG1063

[0562] pEG1063, contained in EG11058, was constructed by replacing theNcoI DNA fragment containing ori 43 from pEG1054 with the isolated NcoIDNA fragment containing ori 43 and the alterations in cry3Bb frompEG1707.

[0563] 5.24.5 pEG1095

[0564] pEG1095, contained in EG11095, was constructed by replacing theMluI-SpeI DNA fragment in pEG1701 with the corresponding MluI-SpeI DNAfragment from pEG1086.

5.25 Example 25

[0565] Primers Utilized in Constructing Cry3Bb* Variants

[0566] Shown below are the primers used for site-directed mutagenesis,triplex PCR™ and quasi-random PCR™ to prepare the cry3Bb* variants asdescribed above. Primers were obtained from Ransom Hill Bioscience, Inc.(Ramona, Calif.) and Integrated DNA Technologies, Inc. (Coralville,Iowa). The specific composition of the primers containing particulardegeneracies at one or more residues is given in Section 5.30, Example30. 5.25.1 PRIMER FW001: 5′-AGACAACTCTACAGTAAAAGATG-3′ (SEQ ID NO:71)5.25.2 PRIMER FW006: 5′-GGTAATTGGTCAATAGAATC-3′ (SEQ ID NO:72) 5.25.3PRIMER MVT095: 5′-CAGAAGATGTTGCTGAATTCNNNCATAGACAATTAAAAC-3′ (SEQ IDNO:73) 5.25.4 PRIMER MVT097: 5′-GATGTTGCTGAATTCTATNNNAGACAATTAAAAC-3′(SEQ ID NO:74) 5.25.5 PRIMER MVT091:5′-CCCATTTTATGATATTBDNTTATACTCAAAAGG-3′ (SEQ ID NO:75) 5.25.6 PRIMERMVT075:5′-AGCTATGCTGGTCTCGGAAGAAAEFNFFNFINJFJFJNFINJFJAAAAGAAGCCAAGATCGAAT-3′(SEQ ID NO:76) 5.25.7 PRIMER MVT076:5′-GGTCACCTAGGTCTCTCTTCCAGGAATTTAACGCATTAAC-3′ (SEQ ID NO:77) 5.25.8PRIMER MVT111:5′-AGCTATGCTGGTCTCCCATTTJEHIEJEJJEIIKRRJEHEIJEENIIIGTTAAAACAGAACTAAC-3′(SEQ ID NO:78) 5.25.9 PRIMER MVT094:5′-ATCCAGTGGGGTCTCAAATGGGAAAAGTACAATTAG-3′ (SEQ ID NO:79) 5.25.10 PRIMERMVT103:5′-CATTTTTACGGATCCAATTTTTJFFFJNEEJEFNFJNFEILEIJEOGGACCAACTTTTTTGAG-3′(SEQ ID NO:80) 5.25.11 PRIMER MYT081:5′-GAATTTCATACGCGTCTTCAACCTGGTJEHJJJIINMEEIEJTCTTTCAATTATTGGTCTGG-3′(SEQ ID NO:81) 5.25.12 PRIMER MVT085:5′-AAAAGTTTATCGAACTATAGCTAATACAGACGTAGCGGCTJQQFFNEEJIIIJEEIGTATATTTAGGTGTTACG-3′(SEQ ID NO:82) 5.25.13 PRIMER A 3B2PFLM1: 5′-GGAGTTCCATTTGCTGGGGC-3′(SEQ ID NO:83) 5.25.14 PRIMER B 3B2PFLM2: 5′-ATCTCCATAAAATGGGG-3′ (SEQID NO:84) 5.25.15 PRIMER C 3B2165DG:5′-GCGAAGTAAAAGAAGCCAAGGTCGAATAAGGG-3′ (SEQ ID NO:86) 5.25.16 PRIMER D3B2160SKRD: 5′-CCTTTAAGTTTGCGAAATCCACACAGCCAAGGTCGAATAAGGG-3′ (SEQ IDNO:86) 5.25.17 PRIMER E 3B2290VP:5′-CCCATTTTATGATGTTCGGTTATACCCAAAAGGGG-3′ (SEQ ID NO:87) 5.25.18 PRIMERF 3B2EDA104: 5′-GGCCAAGTGAAGACCCATGGAAGGC-3′ (SEQ ID NO:88) 5.25.19PRIMER G 3B2KG189: 5′-GCAGTTTCCGGATTCGAAGTGC-3′ (SEQ ID NO:89) 5.25.20PRIMER WD112: 5′-CCGCTACGTCTGTATTA-3′ (SEQ ID NO:90) 5.25.21 PRIMERWD115: 5′-ATAATGGAAGCACCTGA-3′ (SEQ ID NO:91) 5.25.22 PRIMER MVT105:5′-AGCTATGCTGGTCTCTTCTTAEJIFEIIEFFIJFIJIINACAATTCCATTTTTTACTTGG-3′ (SEQID NO:92) 5.25.23 PRIMER MVT092:5′-ATCCAGTTGGGTCTCTAAGAAACAAACCGCGTAATTAAGC-3′ (SEQ ID NO:93) 5.25.24PRIMER MVT070: 5′-CCTCAAGGGTTATAACATCC-3′ (SEQ ID NO:94) 5.25.25 PRIMERMVT083: 5′-GTACAAAAGCTAAGCTTTIEJIINPEEMEEIJNJESCGAACTATAGCTAATACAG-3′(SEQ ID NO:95)

5.26 Example 26

[0567] Sequence Analysis of Altered Cry3Bb Genes

[0568]E. coli DH5α™ (GIBCO BRL, Gaithersburg, Md.), JM110 and Sure™(Stratagene, La Jolla, Calif.) cells were sometimes used amplify plasmidDNA for sequencing. Plasmids were transformed into these cells using themanufacturers' procedures. DNA was sequenced using the Sequenase® 2.0DNA sequencing kit purchased from U.S. Biochemical Corporation(Cleveland, Ohio). The plasmids described in Section 6, their respectivedivergence from WT cry3Bb sequence, the resulting amino acid changes andthe protein structure site of the changes are listed in Table 11. TABLE11 DNA SEQUENCE CHANGES OF CRY3BB* GENES AND RESULTING AMINO ACIDSUBSTITUTIONS OF THE CRY3BB* PROTEINS Structural Site of Plasmid cry3Bb*DNA Sequence Cry3Bb* Amino Acid Sequence Alteration pEG1707 A460T,C461T, A462T, C464A, T465C, T466C, T467A, T154F, P155H, L156H, L158R1α3, 4 A468T, A469T, G470C, T472C, T473G, G474T, A477T, A478T, G479CpEG1708 T687C, T688C, A689T, C691A, A692G Y230L, H231S α6 pEG1709 T667C,T687C, T688A, A689G, C691A, A692G S223P, Y230S α6 pEG1710 T687C, A692GH231R α6 pEG1711 T687C, C691A H231N, T241S α6 pEG1712 T687C, C691A,A692C, T693C H231T α6 pEG1713 C868A, G869A, G870T R290N 1α7, β1 pEG1714C932T, A938C, T942G, G949A, T954C S311L, N313T, E317K 1β1, α8 pEG1715T931A, A933C, T942A, T945A, G949A, A953G, S311T, E317K, Y318C 1β1, α8T954C pEG1716 T931G, A933C, C934G, T945G, C946T, A947G, S311A, L312V,Q316W 1β1, α8 G951A, T954C pEG1717 T687C, A692G, C932T, A938C, T942G,G949A, H231R, S311L, N313T, E317K α6, 1β1, α8 T954C pEG1718 T931A,A933G, T935C, T936A, A938C, T939C, S311T, L312P, N313T, E317N 1β1, α8T942C, T945A, G951T, T954C pEG1719 T931G, A933C, T936G, T942C, C943T,T945A, S311A, Q316D 1β1, α8 C946G, G948C, T954C pEG1720 T861C, T866C,C868A, T871C, T872G, A875T, I289T, L291R, Y292F, S293R 1α7, β1 T877A,C878G, A882G pEG1721 T687C, A692G, C932T H231R, S311L α6, 1β1, α8pEG1722 T931A, C932T, A933C, T936C, T942G, T945A, T954C S311I 1β1, α8pEG1723 T931A, C932T, A933C, T936C, A937G, A938T, S311I, N313H 1β1, α8C941A, T942C, T945A, C946A, A947T, A950T, T954C pEG1724 A933C, T936C,A937G, A938T, C941A, T942C, N313V, T314N, Q316M, E317V 1β1, α8 T945A,C946A, A947T, A950T, T954C pEG1725 A933T, A938G, T939G, T942A, T944C,T945A, N313R, L315P, Q316L, E317A 1β1, α8 A947T, G948T, A950C, T954CpEG1726 A860T, T861C, G862A, C868T, G869T, T871C, Y287F, D288N, R290L1α7, β1 A873T, T877A, C878G, A879T pEG1727 C868G, G869T R290V 1α7, β1pEG1041 A494G D165G α4 pEG1046 G479A, A481C, A482C, A484C, G485A, S160N,K161P, P162H, D165G α4 A486C, A494G pEG1047 A865G, T877C I289V, S293P1α7, β1 pEG1052 G479A, A481C, A482C, A484C, G485A, A486C, S160N, K161P,P162H, D165G, α4, 1α7, β1 A494G, A865G, T877C I289V, S293P pEG1054T309A, Δ310, Δ311, Δ312 D103E, ΔA104 1α2a, 2b pEG1057 A565G, A566G K189G1α4, 5 pEG1062 T309A, Δ310, Δ311, Δ312, G479A, A481C, A482C, D103E,ΔA104, S160N, K161P, 1α2a, 2b α4 A484C, G485A, A486C, A494G P162H, D165GpEG1063 T309A, Δ310, Δ311, Δ312, A460T, C461T, A462T, D103E, ΔA104,T154F, P155H, 1α2a, 2b 1α3, 4 C464A, T465C, T466C, T467A, A468T, A469T,L156H, L158R G470C, T472C, T473G, G474T, A477T, A478T, G479C pEG1084A494G, T931A, A933C, T942A, T945A, G949A, D165G, S311T, E317K α4, 1β1,α8 T954C pEG1085 A494G, A865G, T877C, T914C, T931G, A933C, D165G, I289V,S293P, F305S, α4, 1α7, β1, β1, 1β1, α8 C934G, T945G, C946T, A947G,G951A, T954C, S311A, L312V, Q316W, Q348R, β2, β3b A1043G, T1094C V365ApEG1086 A865G, T877C, A1043G I289V, S293P, Q348R 1α7, β1, β2 pEG1087A494G, C932T D165G, S311L α4, 1β1, α8 pEG1095 A1043G Q348R β2

5.27 Example 27

[0569] Expression of Cry3Bb* Proteins

[0570] 5.27.1 Culture Conditions

[0571] LB agar was prepared using a standard formula (Maniatis et al.,1982). Starch agar was obtained from Difco Laboratories (Detroit, Mich.)and supplemented with an additional 5 g/l of agar. C2 liquid medium isdescribed by Donovan et al. (1988). C2 medium was sometimes preparedwithout the phosphate buffer (C2-P). All cultures were incubated at 25°C. to 30° C.; liquid cultures were also shaken at 250 rpm, untilsporulation and lysis had occurred.

[0572] 5.27.2 Transformation Conditions

[0573] pEG1701 and derivatives thereof were introduced intoacrystalliferious B. thuringiensis var. kurstaki EG7566 (Baum, 1994) orEG10368 (U.S. Pat. No. 5,322,687) by the electroporation method ofMacaluso and Mettus (1991). In some cases, the method was modified asfollows to maximize the number of transformants. The recipient B.thuringiensis strain was inoculated from overnight growth at 30° C. onLB agar into brain heart infusion plus 0.5% glycerol, grown to anoptical density of approximately 0.5 at 600 nm, chilled on ice for 10min, washed 2× with EB and resuspended in a 1/50 volume of EB.Transformed cells were selected on LB agar or starch agar plus 5 μg/mlchloramphenicol. Visual screening of colonies was used to identifytransformants producing crystalline protein; those colonies weregenerally more opaque than colonies that did not produce crystallineprotein.

[0574] 5.27.3 Strain and Protein Designations

[0575] A transformant containing an altered cry3Bb* gene encoding analtered Cry3Bb* protein is designated by an “EG” number, e.g., EG11231.The altered Cry3Bb* protein is designated Cry3Bb followed by the strainnumber, e.g., Cry3Bb.11231. Collections of proteins with alterations ata structural site are designated Cry3Bb followed by the structural site,e.g., Cry3Bb.lβ2,3. Table 12 lists the plasmids pertinent to thisinvention, the new B. thuringiensis strains containing the plasmids, theacrystalliferous B. thuringiensis recipient strain used, and theproteins produced by the new strains.

5.28 Example 28

[0576] Generation and Characterization of Cry3Bb-60

[0577] 5.28.1 Generation of Cry3Bb-60

[0578] Cry3Bb-producing strain EG7231 (U.S. Pat. No. 5,187,091) wasgrown in C2 medium plus 3 mg/ml chloramphenicol. Following sporulationand lysis, the culture was washed with water and Cry3Bb protein purifiedby the NaBr solubilization and recrystallization method of Cody et al.(1992). Protein concentration was determined by BCA Protein Assay(Pierce, Rockford, Ill.). Recrystallized protein was solubilized in 10ml of 50 mM KOH per 100 mg of Cry3Bb protein and buffered to pH 9.0 with100 mM CAPS (3-[cyclohexylamino]-1-propanesulfonic acid), pH 9.0. Thesoluble toxin was treated with trypsin at a weight ratio of 50 mg toxinto 1 mg trypsin for 20 min to overnight at room temperature. Trypsincleaves proteins on the carboxyl side of available arginine and lysineresidues. For 8-dose bioassay, the solubilization conditions werealtered slightly to increase the concentration of protein: 50 mM KOH wasadded dropwise to 2.7 ml of a 12.77 mg/ml suspension of purified Cry3Bb*until crystal solubilization occurred. The volume was then adjusted to 7ml with 100 mM CAPS, pH 9.0. TABLE 12 PLASMIDS CARRYING ALTERED CRY3BB*GENES TRANSFORMED INTO B. THURINGIENSIS FOR EXPRESSION OF ALTEREDCRY3BB* PROTEINS Plasmid Designation New BT Strain Expressed ProteinpEG1701 EG11204 WT Cry3Bb pEG1701 EG11037 WT Cry3Bb pEG1707 EG11221Cry3Bb.11221 pEG1708 EG11222 Cry3Bb.11222 pEG1709 EG11223 Cry3Bb.11223pEG1710 EG11224 Cry3Bb.11224 pEG1711 EG11225 Cry3Bb.11225 pEG1712EG11226 Cry3Bb.11226 pEG1713 EG11227 Cry3Bb.11227 pEG1714 EG11228Cry3Bb.11228 pEG1715 EG11229 Cry3Bb.11229 pEG1716 EG11230 Cry3Bb.11230pEG1717 EG11231 Cry3Bb.11231 pEG1718 EG11232 Cry3Bb.11232 pEG1719EG11233 Cry3Bb.11233 pEG1720 EG11234 Cry3Bb.11234 pEG1721 EG11235Cry3Bb.11235 pEG1722 EG11236 Cry3Bb.11236 pEG1723 EG11237 Cry3Bb.11237pEG1724 EG11238 Cry3Bb.11238 pEG1725 EG11239 Cry3Bb.11239 pEG1726EG11241 Cry3Bb.11241 pEG1727 EG11242 Cry3Bb.11242 pEG1041 EG11032Cry3Bb.11032 pEG1046 EG11035 Cry3Bb.11035 pEG1047 EG11036 Cry3Bb.11036pEG1052 EG11046 Cry3Bb.11046 pEG1054 EG11048 Cry3Bb.11048 pEG1057EG11051 Cry3Bb.11051 pEG1062 EG11057 Cry3Bb.11057 pEG1063 EG11058Cry3Bb.11058 pEG1084 EG11081 Cry3Bb.11081 pEG1085 EG11082 Cry3Bb.11082pEG1086 EG11083 Cry3Bb.11083 pEG1087 EG11084 Cry3Bb.11084 pEG1095EG11095 Cry3Bb.11095 pEG1098 EG11098 Cry3Bb.11098 pEG1701.1β2, 3collection of unnamed strains Cry3Bb.1β2, 3 pEG1701.1β6, 7 collection ofunnamed strains Cry3Bb.1β6, 7 pEG1701.1β10, 11 collection of unnamedstrains Cry3Bb.1β10, 11

[0579] 5.28.2 Determination of Molecular Weight of Cry3Bb-60

[0580] The molecular weight of the predominant trypsin digestionfragment of Cry3Bb was determined to be 60 kDa by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis using commercial molecular weightmarkers. This digestion fragment is designated Cry3Bb-60. No furtherdigestion of the 60 kDa cleavage product was observed.

[0581] 5.28.3 Determination of NH₂-Terminus of Cry3Bb-60

[0582] To determine the NH₂-terminal sequence of Cry3Bb-60, the trypsindigest was fractionated by SDS-PAGE and transferred to Immobilon™-Pmembrane (Millipore Corporation, Bedford, Mass.) following standardwestern blotting procedures. After transfer, the membrane was rinsedtwice with water then stained with 0.025% Coomassie Brilliant Blue R-250plus 40% methanol for 5 min, destained with 50% methanol and rinsed inwater. The Cry3Bb.60 band was excised with a razor blade. NH₂-terminalsequencing was performed at the Tufts Medical School, Department ofPhysiology (Boston, Mass.) using standard automated Edman degradationprocedures. The NH₂-terminal amino acid sequence was determined to beSKRSQDR (SEQ ID NO:96), corresponding to amino acids 160-166 of Cry3Bb.Trypsin digestion occurred on the carboxyl side of amino acid R159resulting in the removal of helices 1-3.

5.29 Example 29

[0583] Bioactivity of Cry3Bb* Proteins

[0584] 5.29.1 Culture Conditions and Protein Concentration Determination

[0585] Cultures for 1-dose bioassays were grown in C2-P plus 5 μg/mlchloramphenicol (C2-P/cm5) then diluted with 3 volumes of 0.005% TritonX-100®. The protein concentrations of these cultures were notdetermined. Cultures for 8-dose bioassays were grown in C2/cm5, washed1-2 times with 1-2 volumes of sterile water and resuspended in 1/10volume of sterile 0.005% Triton X-100®. The toxin protein concentrationof each concentrate was determined as described by Brussock and Currier(1990), omitting the treatment with 3 M HEPES. The protein concentrationwas adjusted to 3.2 mg/ml in 0.005% Triton X-100® for the top dose ofthe assay. Cry3Bb.60 was produced and quantified for 8-dose assay asdescribed in Section 9.1.

[0586] 5.29.2 Insect Bioassays

[0587]Diabrotica undecimpunctata howardi Barber (southern corn rootwormor SCRW) and Diabrotica virgifera virgifiera LeConte (western cornrootworm or WCRW) larvae were reared as described by Slaney et al.(1992). Eight-dose assays and probit analyses were performed asdescribed by Slaney et al. (1992). Thirty-two larvae were tested perdose at 50 μl of sample per well of diet (surface area of 175 mm²).Positive controls were WT Cry3Bb-producing strains EG11037 or EG11204.All bioassays were performed using 128-well trays containingapproximately 1 ml of diet per well with perforated mylar sheet covers(C-D International Inc., Pitman, N.J.). One-dose assays were performedessentially the same except only 1 dose was tested per strain. All assaywere replicated at least twice.

[0588] 5.29.3 Insect Bioassay Results: 1Dose Assays Against SCRW

[0589] Results from 1-dose assays are expressed as the relativemortality (RM) of the experimental strain compared to WT (% mortality ofexperimental culture divided by % mortality of WT culture). Altered andimproved Cry3Bb proteins derived from plasmids constructed using PCR™methods introducing random or semi-random changes into the cry3Bb genesequence were distinguished from other altered but not improved Cry3Bbproteins by replicated, 1-dose assay against SCRW larvae. Those proteinsshowing increased activity (defined as RM≧1.5) compared to WT Cry3Bb or,in the case of proteins with combinations of altered sites, compared toa “parental” altered Cry3Bb protein were further characterized by 8-doseassay. The overall RM “pattern” produced by 1-dose assay results from acollection of proteins carrying random or semi-random alterations withina single structural region, e.g., in lβ2,3, can be used to determine ifthat structural region is important for bioactivity. Retention of WTlevels of activity (RM≈1) indicate changes are tolerated in that region.Overall loss of activity (RM<1) distinguishes the region as importantfor bioactivity.

[0590] 5.29.4 Cry3Bb.Lβ2,3: Results of 1-Dose Bioassays Against SCRW

[0591] Cry3Bb.lβ2.3 protein are a collection of proteins altered in thelβ2,3 region of Cry3Bb (see Section 5.3.4). Typical results of 1-doseassays of these altered proteins are shown in FIG. 12. The RM values forCry3Bb.lβ2,3 proteins are less than 1, with a few exceptions of valuesclose to 1, indicating that this region is important for toxicity.

[0592] 5.29.5 Cry3Bb.lβ6,7: Results of 1-Dose Bioassays Against SCRW

[0593] Cry3Bb.lβ6,7 proteins are a collection of proteins altered in thelβ6,7 region of Cry3Bb (see Section 5.3.5). Typical results of 1-doseassays of these altered proteins are shown in FIG. 13. With a fewexceptions of values close to 1, the RM values for Cry3Bb.lβ6,7 proteinsare less than 1, indicating that this region is important for toxicity.

[0594] 5.29.6 Cry3Bb.lβP10,11: Results of 1-Dose Bioassays Against SCRW

[0595] Cry3Bb.lβ10,11 proteins are a collection of proteins altered inthe lβ10,11 region of Cry3Bb (see Section 5.3.6). Typical results of1-dose assays of these altered proteins are shown in FIG. 14. With a fewexceptions of values close to 1, the RM values for Cry3Bb.lβ10,11proteins are less than 1, indicating that this region is important forbioactivity.

[0596] 5.29.7 Insect Bioassay Results: Results of 8-Dose Assays AgainstSCRW

[0597] Results from 8-dose assays are expressed as an LC₅₀ value(protein concentration giving 50% mortality) with 95% confidenceintervals. The LC₅₀ values with 95% confidence intervals of alteredCry3Bb proteins showing improved activities against SCRW larvae and LC₅₀values of the WT Cry3Bb control determined at the same time are listedin Table 13 along with the fold increase over WT activity for eachimproved protein. TABLE 13 DESIGNED CRY3BB PROTEINS WERE TESTED AGAINSTSCRW LARVAE IN REPLICATED, 8-DOSE ASSAYS TO DETERMINE THE LC₅₀ VALUESLC₅₀ μg/well (95% C.I.) WT Cry3Bb Fold Increase Improved ProteinImproved Protein Control Over WT Activity Cry3Bb.60  6.7 (5.3-8.4) 24.1(15-39) 3.6× Cry3Bb.11221  3.2 (2.5-4) 20.5 (14.5-29) 6.4× Cry3Bb.11222 7.3 (6-9) 29.4 (23-37) 4.0× Cry3Bb.11223 10.5 (9-12) 29.4 (23-37) 2.8×Cry3Bb.11224  6.5 (5.1-8.2) 32.5 (25-43) 5.0× Cry3Bb.11225 13.7(11-16.8) 49.5 (39-65) 3.6× Cry3Bb.11226 16.7 (10.6-24.2) 49.5 (39-65)3.0× Cry3Bb.11227 11.1 (9.1-13.5) 21.3 (16-28) 1.9× Cry3Bb.11228  8.0(6.6-9.8) 32.9 (25-45) 4.1× Cry3Bb.11229  7.2 (5.8-8.8) 18.2 (15-22)2.5× Cry3Bb.11230  7.0 (5.8-8.6) 32.9 (25-45) 4.7× Cry3Bb.11231  3.3(3.0-3.7) 26.1 (22-31) 7.9× Cry3Bb.11232  6.4 (5.4-7.7) 32.9 (25-45)5.1× Cry3Bb.11233 15.7 (12-20) 32.9 (25-45) 2.2× Cry3Bb.11234   7 (6-9)  29 (22-39) 4.1× Cry3Bb.11235  4.2 (3.6-4.9) 13.3 (10-17) 3.2×Cry3Bb.11236 11.6 (9-15) 36.4 (27-49) 3.1× Cry3Bb.11237  6.8 (4-11) 36.4(27-49) 5.4× Cry3Bb.11238 13.9 (11-17) 36.4 (27-49) 2.6× Cry3Bb.1123913.0 (10-16) 36.4 (27-49) 2.8× Cry3Bb.11241   11 (7-16)   29 (22-39)2.6× Cry3Bb.11242 11.9 (9.2-16)   30 (23-38) 2.5× Cry3Bb.11032  4.2(3.6-4.9) 13.3 (10-17) 3.1× Cry3Bb.11035 10.3 (8-13) 27.9 (23-34) 2.7×Cry3Bb.11036  6.5 (5.1-7.9) 27.9 (23-34) 4.3× Cry3Bb.11046 12.1 (8-19)31.2 (25-39) 2.6× Cry3Bb.11048  8.3 (6-11) 35.4 (24-53) 4.3×Cry3Bb.11051 11.8 (8-16) 35.4 (24-53) 3.0× Cry3Bb.11057  8.8 (7-11) 29.5(24-36) 3.4× Cry3Bb.11058  9.6 (6-14) 33.4 (27-43) 3.5× Cry3Bb.11081 8.5 (7-11) 51.5 (37-79) 6.1× Cry3Bb.11082 10.6 (8-13) 51.5 (37-79) 4.9×Cry3Bb.11083  7.0 (5-10) 51.5 (37-79) 7.4× Cry3Bb.11084  7.2 (4-12) 51.5(37-79) 7.2× Cry3Bb.11095 11.1 (9-14) 51.5 (37-79) 4.6× Cry3Bb.11098

[0598] 5.29.8 Insect Bioassay Results: 8-Dose Assays Against WCRW

[0599] WCRW larvae are delicate and difficult to work with. Therefore,only some of the designed Cry3Bb showing improved activity against SCRWlarvae were also tested against WCRW larvae in 8-dose assays. The LC₅₀determinations for the designed Cry3Bb proteins are shown in Table 14along with the LC₅₀ values of the WT Cry3Bb control determined at thesame time. TABLE 14 CRY3BB* PROTEINS SHOWING IMPROVED ACTIVITY AGAINSTSCRW LARVAE ALSO SHOW IMPROVED ACTIVITY AGAINST WCRW LARVAE LC₅₀ μg/well(95% C.I.) WT Cry3Bb Fold Increase Improved Protein Improved ProteinControl Over WT Activity EG11083  6.3 (4.7-8.2) 63.5 (46-91) 10.1×EG11230 24.2 (13-40)  4.5 (2.1-7.4) 5.4× EG11231 32.2 (14-67)  2.5(1.7-3.6) 12.9×

5.30 Example 30

[0600] Channel Activity

[0601] Ion channels produced by Cry3Bb and some of its derivatives weremeasured by the methods described by Slatin et al. (1990). In someinstances, lipid bilayers were prepared from a mixture of 4:1phophatidylethanolamine (PE):phosphatidylcholine (PC). Toxin protein wassolubilized from washed, C2 medium, B. thuringiensis cultures with 12 mMKOH. Following centrifugation to remove spores and other debris, 10 μgof soluble toxin protein was added to the cis compartment (4.5 mlvolume) of the membrane chamber. Protein concentration was determinedusing the BCA Protein Assay (Pierce).

[0602] 5.30.1 Channel Activity of WT Cry3Bb.

[0603] Upon exposure to black lipid membranes, Cry3Bb forms ion channelswith various conductance states. The channels formed by Cry3Bb arerarely discrete channels with well resolved open and closed states andusually require incubation of the toxin with the membrane for 30-45 minbefore any channel-like events are observed. After formation of theinitial conductances, the size increases from approximately 200 pS toover 10,000 pS over 2-3 h. Only the small conductances (≦200 pS) arevoltage dependent. Over 200 pS, the conductances are completelysymmetric. Cry3Bb channels also exhibit β-mercaptoethanol-dependentactivation, growing from small channel conductances of ˜200 pS toseveral thousand pS within 2 min of the addition of β-mercaptoethanol tothe cis compartment of the membrane chamber.

[0604] 5.30.2 Cry3Bb.11032

[0605] The channel activity of Cry3Bb.11032 is much like WT Cry3Bb whenthe solubilized toxin protein is added to the cis compartment of themembrane chamber. However, when this protein is artificiallyincorporated into the membrane by forming or “painting” the membrane inthe presence of the Cry3Bb.11032 protein, a 16-fold increase in theinitial channel conductances is observed (˜4000 pS). This phenomenon isnot observed with WT Cry3Bb.

[0606] 5.30.3 Cry3Bb.11035

[0607] Upon exposure to artificial membranes, the Cry3Bb.11035 proteinspontaneously forms channels that grow to large conductances within arelatively short time span (˜5 min). Conductance values ranges from3000-6000 pS and, like WT Cry3Bb, are voltage dependent at lowconductance values.

[0608] 5.30.4 Cry3Bb.11048

[0609] The Cry3Bb.11048 protein is quite different than WT Cry3Bb inthat it appears not to form channels at all, but, rather, formssymmetrical pores with respect to voltage. Once the pore is formed, itremains open and allows a steady conductance ranging from 25 to 130 pS.

[0610] 5.30.5 Cry3Bb.11224 and Cry3Bb.11226

[0611] The metal binding site of WT Cry3Bb formed by H231 in the dimerstructure was removed in proteins Cry3Bb.11224 and Cry3Bb.11226. Theconductances formed by both designed proteins are identical to that ofWT Cry3Bb with the exception that neither of the designed proteinsexhibits β-mercaptoethanol-dependent activation.

[0612] 5.30.6 Cry3Bb.11221

[0613] Cry3Bb.11221 protein has been observed to immediately form smallchannels of 100-200 pS with limited voltage dependence. Some higherconductances were observed at the negative potential. In other studiesthe onset of activity was delayed by 27 min, which is more typical forWT Cry3Bb. Unlike WT Cry3Bb, however, Cry3Bb.11221 forms well resolved,600 pS channels with long open states. The protein eventually reachesconductances of 7000 pS.

[0614] 5.30.7 Cry3Bb.11242

[0615] Cry3Bb.11242 protein forms small conductances immediately uponexposure to an artificial membrane. The conductances grow steadily andrapidly to 6000 pS in approximately 3 min. Some voltage dependence wasnoted with a preference for a negative imposed voltage.

[0616] 5.30.8 Cry3Bb.11230

[0617] Unlike WT Cry3Bb, Cry3Bb.11230 forms well resolved channels withlong open states that do not continue to grow in conductance with time.The maximum observed channel conductances reached 3000 pS. FIG. 15illustrates the difference between the channels formed by Cry3Bb andCry3Bb.11230.

[0618] 5.30.9 Cry3Bb.60

[0619] Cry3Bb.60 forms well resolved ion channels within 20 min ofexposure to an artificial membrane. These channels grow in conductanceand frequency with time. The behavior of Cry3Bb.60 in a planar lipidbilayer differs from Cry3Bb in two significant ways. The conductancescreated by Cry3Bb.60 form more quickly than Cry3Bb and, unlike Cry3Bb,the conductances are stable, having well resolved open and closed statesdefinitive of stable ion channels (FIG. 16).

5.31 Example 31

[0620] Primer Compositions TABLE 15 SEQ ID NO:83 % of Nucleotide inmixture Code A T G C N 25 25 25 25

[0621] TABLE 16 SEQ ID NO:84 % of Nucleotide in mixture Code A T G C N25 25 25 25

[0622] TABLE 17 SEQ ID NO:85 % of Nucleotide in mixture Code A T G C B16 16 52 16 D 70 10 10 10 N 25 25 25 25

[0623] TABLE 18 SEQ ID NO:86 % of Nucleotide in mixture Code A T G C E82 6 6 6 F 6 6 6 82 J 6 82 6 6 I 6 6 82 6 N 25 25 25 25

[0624] TABLE 19 SEQ ID NO:88 % of Nucleotide in mixture Code A T G C J 682 6 6 E 82 6 6 6 H 1 1 1 97 I 6 6 82 6 K 15 15 15 55 R 15 55 15 15

[0625] TABLE 20 SEQ ID NO:90 % of Nucleotide in mixture Code A T G C J 682 6 6 F 6 6 6 82 N 25 25 25 25 E 82 6 6 6 I 6 6 82 6 L 8 1 83 8 O 1 1 197

[0626] TABLE 21 SEQ ID NO:91 % of Nucleotide in mixture Code A T G C J 682 6 6 E 82 6 6 6 H 1 1 1 97 I 6 6 82 6 N 25 25 25 25 M 82 2 8 8

[0627] TABLE 22 SEQ ID NO:92 % of Nucleotide in mixture Code A T G C J 682 6 6 Q 0 9 82 9 F 6 6 6 82 N 25 25 25 25 E 82 6 6 6 I 6 6 82 6

[0628] TABLE 23 SEQ ID NO:92 % of Nucleotide in mixture Code A T G C J 682 6 6 F 6 6 6 82 N 25 25 25 25 E 82 6 6 6 I 6 6 82 6

[0629] TABLE 24 SEQ ID NO:95 % of Nucleotide in mixture Code A T G C J 682 6 6 N 25 25 25 25 E 82 6 6 6 I 6 6 82 6 M 82 2 8 8 P 8 2 8 82 S 1 971 1

5.32 Example 32

[0630] Atomic Coordinates for Cry3Bb

[0631] The atomic coordinates of the Cry3Bb protein are given in theAppendix included in Section 9.1

5.33 Example 33

[0632] Atomic Coordinates for Cry3A

[0633] The atomic coordinates of the Cry3A protein are given in theAppendix included in Section 9.2

5.34 Example 34

[0634] Modification of Cry Genes for Expression in Plants

[0635] Wild-type cry genes are known to be expressed poorly in plants asa full length gene or as a truncated gene. Typically, the G+C content ofa cry gene is low (37%) and often contains many A+T rich regions,potential polyadenylation sites and numerous ATTTA sequences. Table 25shows a list of potential polyadenylation sequences which should beavoided when preparing the “plantized” gene construct. TABLE 25 LIST OFSEQUENCES OF THE POTENTIAL POLYADENYLATION SIGNALS AATAAA* AAGCATAATAAT* ATTAAT AACCAA ATACAT ATATAA AAAATA AATCAA ATTAAA** ATACTAAATTAA** ATAAAA AATACA** ATGAAA CATAAA**

[0636] The regions for mutagenesis may be selected in the followingmanner. All regions of the DNA sequence of the cry gene are identifiedwhich contained five or more consecutive base pairs which were A or T.These were ranked in terms of length and highest percentage of A+T inthe surrounding sequence over a 20-30 base pair region. The DNA isanalysed for regions which might contain polyadenylation sites or ATTTAsequences. Oligonucleotides are then designed which maximize theelimination of A+T consecutive regions which contained one or morepolyadenylation sites or ATTTA sequences. Two potential plantpolyadenylation sites have been shown to be more critical based onpublished reports. Codons are selected which increase G+C content, butdo not generate restriction sites for enzymes useful for cloning andassembly of the modified gene (e.g, BamHI, BglII, SacI, NcoI, EcoRV,etc.). Likewise condons are avoided which contain the doublets TA or GCwhich have been reported to be infrequently-found codons in plants.

[0637] Although the CaMV35S promoter is generally a high levelconstitutive promoter in most plant tissues, the expression level ofgenes driven the CaMV35S promoter is low in floral tissue relative tothe levels seen in leaf tissue. Because the economically importanttargets damaged by some insects are the floral parts or derived fromfloral parts (e.g., cotton squares and bolls, tobacco buds, tomato budsand fruit), it is often advantageous to increase the expression ofcrystal proteins in these tissues over that obtained with the CaMV35Spromotor.

[0638] The 35S promoter of Figwort Mosaic Virus (FMV) is analogous tothe CaMV35S promoter. This promoter has been isolated and engineeredinto a plant transformation vector. Relative to the CaMV promoter, theFMV 35S promoter is highly expressed in the floral tissue, while stillproviding similar high levels of gene expression in other tissues suchas leaf. A plant transformation vector, may be constructed in which thefull length synthetic cry gene is driven by the FMV 35S promoter.Tobacco plants may be transformed with the vector and compared forexpression of the crystal protein by Western blot or ELISA immunoassayin leaf and floral tissue. The FMV promoter has been used to producerelatively high levels of crystal protein in floral tissue compared tothe CaMV promoter.

5.35 Example 35

[0639] Expression of Synthetic Cry Genes with ssRUBISCO Promoters andChloroplast Transit Peptides

[0640] The genes in plants encoding the small subunit of RUBISCO (SSU)are often highly expressed, light regulated and sometimes show tissuespecificity. These expression properties are largely due to the promotersequences of these genes. It has been possible to use SSU promoters toexpress heterologous genes in transformed plants. Typically a plant willcontain multiple SSU genes, and the expression levels and tissuespecificity of different SSU genes will be different. The SSU proteinsare encoded in the nucleus and synthesized in the cytoplasm asprecursors that contain an N-terminal extension known as the chloroplasttransit peptide (CTP). The CTP directs the precursor to the chloroplastand promotes the uptake of the SSU protein into the chloroplast. In thisprocess, the CTP is cleaved from the SSU protein. These CTP sequenceshave been used to direct heterologous proteins into chloroplasts oftransformed plants.

[0641] The SSU promoters might have several advantages for expression ofheterologous genes in plants. Some SSU promoters are very highlyexpressed and could give rise to expression levels as high or higherthan those observed with the CaMV35S promoter. The tissue distributionof expression from SSU promoters is different from that of the CaMV35Spromoter, so for control of some insect pests, it may be advantageous todirect the expression of crystal proteins to those cells in which SSU ismost highly expressed. For example, although relatively constitutive, inthe leaf the CaMV35S promoter is more highly expressed in vasculartissue than in some other parts of the leaf, while most SSU promotersare most highly expressed in the mesophyll cells of the leaf. Some SSUpromoters also are more highly tissue specific, so it could be possibleto utilize a specific SSU promoter to express the protein of the presentinvention in only a subset of plant tissues, if for example expressionof such a protein in certain cells was found to be deleterious to thosecells. For example, for control of Colorado potato beetle in potato, itmay be advantageous to use SSU promoters to direct crystal proteinexpression to the leaves but not to the edible tubers.

[0642] Utilizing SSU CTP sequences to localize crystal proteins to thechloroplast might also be advantageous. Localization of the B.thuringiensis crystal proteins to the chloroplast could protect thesefrom proteases found in the cytoplasm. This could stabilize the proteinsand lead to higher levels of accumulation of active toxin. cry genescontaining the CTP could be used in combination with the SSU promoter orwith other promoters such as CaMV35S.

5.36 Example 36

[0643] Targeting of Cry* Proteins to the Extracellular Space or VacuoleThrough the Use of Signal Peptides

[0644] The B. thuringiensis proteins produced from the synthetic genesdescribed here are localized to the cytoplasm of the plant cell, andthis cytoplasmic localization results in plants that are insecticidallyeffective. It may be advantageous for some purposes to direct the B.thuringiensis proteins to other compartments of the plant cell.Localizing B. thuringiensis proteins in compartments other than thecytoplasm may result in less exposure of the B. thuringiensis proteinsto cytoplasmic proteases leading to greater accumulation of the proteinyielding enhanced insecticidal activity. Extracellular localizationcould lead to more efficient exposure of certain insects to the B.thuringiensis proteins leading to greater efficacy. If a B.thuringiensis protein were found to be deleterious to plant cellfunction, then localization to a noncytoplasmic compartment couldprotect these cells from the protein.

[0645] In plants as well as other eukaryotes, proteins that are destinedto be localized either extracellularly or in several specificcompartments are typically synthesized with an N-terminal amino acidextension known as the signal peptide. This signal peptide directs theprotein to enter the compartmentalization pathway, and it is typicallycleaved from the mature protein as an early step incompartmentalization. For an extracellular protein, the secretorypathway typically involves cotranslational insertion into theendoplasmic reticulum with cleavage of the signal peptide occurring atthis stage. The mature protein then passes through the Golgi body intovesicles that fuse with the plasma membrane thus releasing the proteininto the extracellular space. Proteins destined for other compartmentsfollow a similar pathway. For example, proteins that are destined forthe endoplasmic reticulum or the Golgi body follow this scheme, but theyare specifically retained in the appropriate compartment. In plants,some proteins are also targeted to the vacuole, another membrane boundcompartment in the cytoplasm of many plant cells. Vacuole targetedproteins diverge from the above pathway at the Golgi body where theyenter vesicles that fuse with the vacuole.

[0646] A common feature of this protein targeting is the signal peptidethat initiates the compartmentalization process. Fusing a signal peptideto a protein will in many cases lead to the targeting of that protein tothe endoplasmic reticulum. The efficiency of this step may depend on thesequence of the mature protein itself as well. The signals that direct aprotein to a specific compartment rather than to the extracellular spaceare not as clearly defined. It appears that many of the signals thatdirect the protein to specific compartments are contained within theamino acid sequence of the mature protein. This has been shown for somevacuole targeted proteins, but it is not yet possible to define thesesequences precisely. It appears that secretion into the extracellularspace is the “default” pathway for a protein that contains a signalsequence but no other compartmentalization signals. Thus, a strategy todirect B. thuringiensis proteins out of the cytoplasm is to fuse thegenes for synthetic B. thuringiensis genes to DNA sequences encodingknown plant signal peptides. These fusion genes will give rise to B.thuringiensis proteins that enter the secretory pathway, and lead toextracellular secretion or targeting to the vacuole or othercompartments. Signal sequences for several plant genes have beendescribed. One such sequence is for the tobacco pathogenesis relatedprotein PR1b has been previously described (Cornelissen et al., 1986).The PR1b protein is normally localized to the extracellular space.Another type of signal peptide is contained on seed storage proteins oflegumes. These proteins are localized to the protein body of seeds,which is a vacuole like compartment found in seeds. A signal peptide DNAsequence for the β-subunit of the 7S storage protein of common bean(Phaseolus vulgaris), PvuB has been described (Doyle et al., 1986).Based on the published these published sequences, genes may besynthesized chemically using oligonucleotides that encode the signalpeptides for PR1b and PvuB. In some cases to achieve secretion orcompartmentalization of heterologous proteins, it may be necessary toinclude some amino acid sequence beyond the normal cleavage site of thesignal peptide. This may be necessary to insure proper cleavage of thesignal peptide.

5.37 Example 37

[0647] Isolation of Transgenic Maize Resistant to Diabrotics spp. UsingCry3Bb Variants

[0648] 5.37.1 Plant Gene Construction

[0649] The expression of a plant gene which exists in double-strandedDNA form involves transcription of messenger RNA (mRNA) from one strandof the DNA by RNA polymerase enzyme, and the subsequent processing ofthe mRNA primary transcript inside the nucleus. This processing involvesa 3′ non-translated region which adds polyadenylate nucleotides to the3′ end of the RNA. Transcription of DNA into mRNA is regulated by aregion of DNA usually referred to as the “promoter”. The promoter regioncontains a sequence of bases that signals RNA polymerase to associatewith the DNA and to initiate the transcription of mRNA using one of theDNA strands as a template to make a corresponding strand of RNA.

[0650] A number of promoters which are active in plant cells have beendescribed in the literature. Such promoters may be obtained from plantsor plant viruses and include, but are not limited to, the nopalinesynthase (NOS) and octopine synthase (OCS) promoters (which are carriedon tumor-inducing plasmids of Agrobacterium tumefaciens), thecauliflower mosaic virus (CaMV) 19S and 35S promoters, thelight-inducible promoter from the small subunit of ribulose1,5-bisphosphate carboxylase (ssRUBISCO a very abundant plantpolypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter. All ofthese promoters have been used to create various types of DNA constructswhich have been expressed in plants (see e.g., U.S. Pat. No. 5,463,175,specifically incorporated herein by reference).

[0651] The particular promoter selected should be capable of causingsufficient expression of the enzyme coding sequence to result in theproduction of an effective amount of protein. One set of preferredpromoters are constitutive promoters such as the CaMV35S or FMV35Spromoters that yield high levels of expression in most plant organs(U.S. Pat. No. 5,378,619, specifically incorporated herein byreference). Another set of preferred promotors are root enhanced orspecific promoters such as the CaMV derived 4 as-1 promoter or the wheatPOX1 promoter (U.S. Pat. No. 5,023,179, specifically incorporated hereinby reference; Hertig et al., 1991). The root enhanced or specificpromoters would be particularly preferred for the control of cornrootworm (Diabroticus spp.) in transgenic corn plants.

[0652] The promoters used in the DNA constructs (i.e. chimeric plantgenes) of the present invention may be modified, if desired, to affecttheir control characteristics. For example, the CaMV35S promoter may beligated to the portion of the ssRUBISCO gene that represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The resulting chimericpromoter may be used as described herein. For purposes of thisdescription, the phrase “CaMV35S” promoter thus includes variations ofCaMV35S promoter, e.g., promoters derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters may be altered to contain multiple “enhancer sequences” toassist in elevating gene expression.

[0653] The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNA's, fromsuitable eucaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence.

[0654] For optimized expression in monocotyledenous plants such asmaize, an intron should also be included in the DNA expressionconstruct. This intron would typically be placed near the 5′ end of themRNA in untranslated sequence. This intron could be obtained from, butnot limited to, a set of introns consisting of the maize hsp70 intron(U.S. Pat. No. 5,424,412; specifically incorporated herein by reference)or the rice Act1 intron (McElroy et al., 1990). As shown below, themaize hsp70 intron is useful in the present invention.

[0655] As noted above, the 3′ non-translated region of the chimericplant genes of the present invention contains a polyadenylation signalwhich functions in plants to cause the addition of adenylate nucleotidesto the 3′ end of the RNA. Examples of preferred 3′ regions are (1) the3′ transcribed, non-translated regions containing the polyadenylatesignal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as thenopaline synthase (NOS) gene and (2) plant genes such as the peassRUBISCO E9 gene (Fischhoff et al., 1987).

[0656] 5.37.2 Plant Transformation and Expression

[0657] A chimeric plant gene containing a structural coding sequence ofthe present invention can be inserted into the genome of a plant by anysuitable method. Suitable plant transformation vectors include thosederived from a Ti plasmid of Agrobacterium tumefaciens, as well as thosedisclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985)and Eur. Pat. Appl. Publ. No. EP0120516. In addition to planttransformation vectors derived from the Ti or root-inducing (Ri)plasmids of Agrobacterium, alternative methods can be used to insert theDNA constructs of this invention into plant cells. Such methods mayinvolve, for example, the use of liposomes, electroporation, chemicalsthat increase free DNA uptake, free DNA delivery via microprojectilebombardment, and transformation using viruses or pollen (Fromm et al.,1986; Armstrong et al., 1990; Fromm et al., 1990).

[0658] 5.37.3 Construction of Monocot Plant Expression Vectors forCry3Bb Variants

[0659] 5.37.3.1 Design of Cry3Bb Variant Genes for Plant Expression

[0660] For efficient expression of the cry3Bb variants in transgenicplants, the gene encoding the variants must have a suitable sequencecomposition (Diehn et al, 1996). One example of such a sequence is shownfor the v11231 gene (SEQ ID NO:99) which encodes the Cry3Bb11231 variantprotein (SEQ ID NO:100) with Diabrotica activity. This gene was derivedvia mutagenesis (Kunkel, 1985) of a cry3Bb synthetic gene (SEQ IDNO:101) encoding a protein essentially homologous to the protein encodedby the native cry3Bb gene (Gen Bank Accession Number m89794, SEQ IDNO:102). The following oligonucleotides were used in the mutagenesis ofthe original cry3Bb synthetic gene (SEQ ID NO:101) to create the v11231gene (SEQ ID NO:99): Oligo #1: 5′-TAGGCCTCCATCCATGGCAAACCCTAACAATC-3′(SEQ ID NO:103) Oligo #2:5′-TCCCATCTTCCTACTTACGACCCTGCAGAAATACGGTCCAAC-3′ (SEQ ID NO:104) Oligo#3: 5′-GACCTCACCTACCAAACATTCGATCTTG-3′ (SEQ ID NO:105) Oligo #4:5′-CGAGTTCTACCGTAGGCAGCTCAAG-3′ (SEQ ID NO:106)

[0661] 5.37.3.2 Construction of Cry3Bb Monocot Plant Expression Vector

[0662] To place the cry3Bb variant gene v11231 in a vector suitable forexpression in monocotyledonous plants (i.e. under control of theenhanced Cauliflower Mosaic Virus 35S promoter and link to the hsp70intron followed by a nopaline synthase polyadenylation site as in U.S.Pat. No. 5,424,412, specifically incorporated herein by reference), thevector pMON19469 was digested with NcoI and EcoRI. The larger vectorband of approximately 4.6 kb was electrophoresed, purified, and ligatedwith T4 DNA ligase to the NcoI-EcoRI fragment of approximately 2 kbcontaining the v11231 gene (SEQ ID NO:99). The ligation mix wastransformed into E. coli, carbenicillin resistant colonies recovered andplasmid DNA recovered by DNA miniprep procedures. This DNA was subjectedto restriction endonuclease analysis with enzymes such as NcoI and EcoRI(together) NotI, and PstI to identify clones containing pMON33708 (thev11231 coding sequence fused to the hsp70 intron under control of theenhanced CaMV35S promoter).

[0663] To place the v11231 gene in a vector suitable for recovery ofstably transformed and insect resistant plants, the 3.75-kb NotIrestriction fragment from pMON33708 containing the lysine oxidase codingsequence fused to the hsp70 intron under control of the enhanced CaMV35Spromoter was isolated by gel electrophoresis and purification. Thisfragment was ligated with pMON30460 treated with NotI and calfintestinal alkaline phosphatase (pMON30460 contains the neomycinphosphotransferase coding sequence under control of the CaMV35Spromoter). Kanamycin resistant colonies were obtained by transformationof this ligation mix into E. coli and colonies containing pMON33710identified by restriction endonuclease digestion of plasmid miniprepDNAs. Restriction enzymes such as NotI, EcoRV, HindIII, NcoI, EcoRI, andBglII can be used to identify the appropriate clones containing the NotIfragment of pMON33708 in the NotI site of pMON30460 (i.e. pMON33710) inthe orientation such that both genes are in tandem (i.e. the 3′ end ofthe v11231 expression cassette is linked to the 5′ end of the nptIIexpression cassette). Expression of the v11231 protein by pMON33710 incorn protoplasts was confirmed by electroporation of pMON33710 DNA intoprotoplasts followed by protein blot and ELISA analysis. This vector canbe introduced into the genomic DNA of corn embryos by particle gunbombardment followed by paromomycin selection to obtain corn plantsexpressing the v11231 gene essentially as described in U.S. Pat. No.5,424,412, specifically incorporated herein by reference.

[0664] In this example, the vector was introduced via cobombardment witha hygromycin resistance conferring plasmid into immature embryo scutella(IES) of maize, followed by hygromycin selection, and regeneration.Transgenic corn lines expressing the v11231 protein were identified byELISA analysis. Progeny seed from these events were subsequently testedfor protection from Diabrotica feeding.

[0665] 5.37.3.3 In Planta Performance of Cry3Bb.11231

[0666] Transformed corn plants expressing Cry3Bb.11231 protein werechallenged with western corn rootworm (WCR) larvae in both a seedlingand 10 inch pot assay. The transformed genotype was A634, where theprogeny of the R0 cross by A634 was evaluated. Observations includedeffect on larval development (weight), root damage rating (RDR), andprotein expression. The transformation vector containing the cry3Bb genewas pMON33710. Treatments included the positive and negativeiso-populations for each event and an A634 check.

[0667] The seedling assay consisted of the following steps: (i) singleseeds were placed in 1 oz cups containing potting soil; (ii) at spiking,each seedling was infested with 4 neonate larvae; and (iii) afterinfestation, seedlings were incubated for 7 days at 25° C., 50% RH, and14:10 (L:D) photo period. Adequate moisture was added to the pottingsoil during the incubation period to maintain seedling vigor.

[0668] The 10 inch pot assay consisted of the following steps: (i)single seeds were placed in 10 inch pots containing potting soil; (ii)at 14 days post planting, each pot was infested with 800 eggs which havebeen pre-incubated such that hatch would occur 5-7 days postinfestation; and (iii) after infestation, plants were incubated for 4weeks under the same environmental conditions as the seedling assay.Pots were both sub and top irrigated daily.

[0669] For the seedling assay, on day 7 plants were given a root damagerating, and surviving larvae were weighed. Also at this time, Cry3Bbprotein concentrations in the roots were determined by ELISA. The scaleused for the seedling assay to assess root damage is as follows: RDR(root damage rating) 0=no visible feeding; RDR 1=very light feeding; RDR2=light feeding; RDR 3=moderate feeding; RDR 4=heavy feeding; and RDR5=very heavy feeding.

[0670] Results of the seedling assay are shown in Table 26. Plantsexpressing Cry3Bb protein were completely protected by WCR feeding,where surviving larvae within this treatment had not grown. Mean larvalweights ranged from 2.03-2.73 mg for the nonexpressing treatments, wherethe surviving larval average weight was 0.11 mg on the expressing cry3Bbtreatment. Root damage ratings were 3.86 and 0.33 for the nonexpressingand expressing isopopulations, respectively. Larval survival ranged from75-85% for the negative and check treatments, where only 25% of thelarvae survived on the Cry3Bb treatment. TABLE 26 EFFECT OF CRY3BBEXPRESSING PLANTS ON WCR LARVAE IN A SEEDLING ASSAY Plants Larvae Root %Mean ± SD Event Treatment N (ppm) RDR ± SD N Surv Wt. (mg) 16 Negative 70.0 3.86 ± 0.65 21 75 2.73 ± 1.67 16 Positive 3 29.01 0.33 ± 0.45  3 250.11 ± 0.07 A634 Check 4 0.0 — 13 81 2.03 ± 0.83

[0671] For the 10 inch pot assay, at 4 weeks post infestation plantheight was recorded and a root damage rating (Iowa 1-6 scale; Hills andPeters, 1971) was given.

[0672] Results of the 10 inch pot assay are shown in Table 27. Plantsexpressing Cry3Bb protein had significantly less feeding damage and weretaller than the non-expressing plants. Event 16, the higher of the twoexpressing events provided nearly complete control. The negativetreatments had very high root damage ratings indicating very high insectpressure. The positive mean root damage ratings were 3.4 and 2.2 forevent 6 and 16, respectively. Mean RDR for the negative treatment was5.0 and 5.6. TABLE 27 EFFECT OF CRY3BB EXPRESSING CORN IN CONTROLLINGWCR LARVAL FEEDING IN A 10 INCH POT ASSAY Root Plant Event Treatment N(ppm) RDR ± SD Height (cm)  6 Negative 7 0.0 5.0 ± 1.41 49.7 ± 18.72  6Positive 5 7.0 3.4 ± 1.14 73.9 ± 8.67 16 Negative 5 0.0 5.6 ± 0.89 61.2± 7.75 16 Positive 5 55.0 2.2 ± 0.84 83.8 ± 7.15

[0673] In summary, corn plants expressing Cry3Bb protein have asignificant biological effect on WCR larval development as seen in theseedling assay. When challenged with very high infestation levels,plants expressing the Cry3Bb protein were protected from WCR larvalfeeding damage as illustrated in the 10 inch pot assay.

[0674] 6.0 Brief Description of the Sequence Identifiers

[0675] SEQ ID NO:1 DNA sequence of cry3Bb.11221 gene.

[0676] SEQ ID NO:2 Amino acid sequence of Cry3Bb.11221 polypeptide.

[0677] SEQ ID NO:3 DNA sequence of cry3Bb.11222 gene.

[0678] SEQ ID NO:4 Amino acid sequence of Cry3Bb.11222 polypeptide.

[0679] SEQ ID NO:5 DNA sequence of cry3Bb.11223 gene.

[0680] SEQ ID NO:6 Amino acid sequence of Cry3Bb.11223 polypeptide.

[0681] SEQ ID.NO:7 DNA sequence of cry3Bb.11224 gene.

[0682] SEQ ID NO:8 Amino acid sequence of Cry3Bb.11224 polypeptide.

[0683] SEQ ID NO:9 DNA sequence of cry3Bb.11225 gene.

[0684] SEQ ID NO:10 Amino acid sequence of Cry3Bb.11225 polypeptide.

[0685] SEQ ID NO:11 DNA sequence of cry3Bb.11226 gene.

[0686] SEQ ID NO:12 Amino acid sequence of Cry3Bb.11226 polypeptide.

[0687] SEQ ID NO:13 DNA sequence of cry3Bb.11227 gene.

[0688] SEQ ID NO:14 Amino acid sequence of Cry3Bb.11227 polypeptide.

[0689] SEQ ID NO:15 DNA sequence of cry3Bb.11228 gene.

[0690] SEQ ID NO:16 Amino acid sequence of Cry3Bb.11228 polypeptide.

[0691] SEQ ID NO:17 DNA sequence of cry3Bb.11229 gene.

[0692] SEQ ID NO:18 Amino acid sequence of Cry3Bb.11229 polypeptide.

[0693] SEQ ID NO:19 DNA sequence of cry3Bb.11230 gene.

[0694] SEQ ID NO:20 Amino acid sequence of Cry3Bb.11230 polypeptide.

[0695] SEQ ID NO:21 DNA sequence of cry3Bb.11231 gene.

[0696] SEQ ID NO:22 Amino acid sequence of Cry3Bb.11231 polypeptide.

[0697] SEQ ID NO:23 DNA sequence of cry3Bb.11232 gene.

[0698] SEQ ID NO:24 Amino acid sequence of Cry3Bb.11232 polypeptide.

[0699] SEQ ID NO:25 DNA sequence of cry3Bb.11233 gene.

[0700] SEQ ID NO:26 Amino acid sequence of Cry3Bb.11233 polypeptide.

[0701] SEQ ID NO:27 DNA sequence of cry3Bb.1234 gene.

[0702] SEQ ID NO:28 Amino acid sequence of Cry3Bb.11234 polypeptide.

[0703] SEQ ID NO:29 DNA sequence of cry3Bb.11235 gene.

[0704] SEQ ID NO:30 Amino acid sequence of Cry3Bb.11235 polypeptide.

[0705] SEQ ID NO:31 DNA sequence of cry3Bb.11236 gene.

[0706] SEQ ID NO:32 Amino acid sequence of Cry3Bb.11236 polypeptide.

[0707] SEQ ID NO:33 DNA sequence of cry3Bb.11237 gene.

[0708] SEQ ID NO:34 Amino acid sequence of Cry3Bb.11237 polypeptide.

[0709] SEQ ID NO:35 DNA sequence of cry3Bb.11238 gene.

[0710] SEQ ID NO:36 Amino acid sequence of Cry3Bb.11238 polypeptide.

[0711] SEQ ID NO:37 DNA sequence of cry3Bb.11239 gene.

[0712] SEQ ID NO:38 Amino acid sequence of Cry3Bb.11239 polypeptide.

[0713] SEQ ID NO:39 DNA sequence of cry3Bb.11241 gene.

[0714] SEQ ID NO:40 Amino acid sequence of Cry3Bb.11241 polypeptide.

[0715] SEQ ID NO:41 DNA sequence of cry3Bb.11242 gene.

[0716] SEQ ID NO:42 Amino acid sequence of Cry3Bb.11242 polypeptide.

[0717] SEQ ID NO:43 DNA sequence of cry3Bb.11032 gene.

[0718] SEQ ID NO:44 Amino acid sequence of Cry3Bb.11032 polypeptide.

[0719] SEQ ID NO:45 DNA sequence of cry3Bb.11035 gene.

[0720] SEQ ID NO:46 Amino acid sequence of Cry3Bb.11035 polypeptide.

[0721] SEQ ID NO:47 DNA sequence of cry3Bb.11036 gene.

[0722] SEQ ID NO:48 Amino acid sequence of Cry3Bb.11036 polypeptide.

[0723] SEQ ID NO:49 DNA sequence of cry3Bb.11046 gene.

[0724] SEQ ID NO:50 Amino acid sequence of Cry3Bb.11046 polypeptide.

[0725] SEQ ID NO:51 DNA sequence of cry3Bb.11048 gene.

[0726] SEQ ID NO:52 Amino acid sequence of Cry3Bb.11048 polypeptide.

[0727] SEQ ID NO:53 DNA sequence of cry3Bb.11051 gene.

[0728] SEQ ID NO:54 Amino acid sequence of Cry3Bb.11051 polypeptide.

[0729] SEQ ID NO:55 DNA sequence of cry3Bb.11057 gene.

[0730] SEQ ID NO:56 Amino acid sequence of Cry3Bb.11057 polypeptide.

[0731] SEQ ID NO:57 DNA sequence of cry3Bb.11058 gene.

[0732] SEQ ID NO:58 Amino acid sequence of Cry3Bb.11058 polypeptide.

[0733] SEQ ID NO:59 DNA sequence of cry3Bb.11081 gene.

[0734] SEQ ID NO:60 Amino acid sequence of Cry3Bb.11081 polypeptide.

[0735] SEQ ID NO:61 DNA sequence of cry3Bb.11082 gene.

[0736] SEQ ID NO:62 Amino acid sequence of Cry3Bb.11082 polypeptide.

[0737] SEQ ID NO:63 DNA sequence of cry3Bb.11083 gene.

[0738] SEQ ID NO:64 Amino acid sequence of Cry3Bb.11083 polypeptide.

[0739] SEQ ID NO:65 DNA sequence of cry3Bb. 11084 gene.

[0740] SEQ ID NO:66 Amino acid sequence of Cry3Bb.11084 polypeptide.

[0741] SEQ ID NO:67 DNA sequence of cry3Bb.11095 gene.

[0742] SEQ ID NO:68 Amino acid sequence of Cry3Bb.11095 polypeptide.

[0743] SEQ ID NO:69 DNA sequence of cry3Bb.60 gene.

[0744] SEQ ID NO:70 Amino acid sequence of Cry3Bb.60 polypeptide.

[0745] SEQ ID NO:71 Primer FW001.

[0746] SEQ ID NO:72 Primer FW006.

[0747] SEQ ID NO:73 Primer MVT095.

[0748] SEQ ID NO:74 Primer MVT097.

[0749] SEQ ID NO:75 Primer MVT091.

[0750] SEQ ID NO:76 Primer MVT075.

[0751] SEQ ID NO:77 Primer MVT076.

[0752] SEQ ID NO:78 Primer MVT111.

[0753] SEQ ID NO:79 Primer MVT094.

[0754] SEQ ID NO:80 Primer MVT103.

[0755] SEQ ID NO:81 Primer MVT081.

[0756] SEQ ID NO:82 Primer MVT085.

[0757] SEQ ID NO:83 Primer A.

[0758] SEQ ID NO:84 Primer B.

[0759] SEQ ID NO:85 Primer C.

[0760] SEQ ID NO:86 Primer D.

[0761] SEQ ID NO:87 Primer E.

[0762] SEQ ID NO:88 Primer F.

[0763] SEQ ID NO:89 Primer G.

[0764] SEQ ID NO:90 Primer WD112.

[0765] SEQ ID NO:91 Primer WD115.

[0766] SEQ ID NO:92 Primer MVT105.

[0767] SEQ ID NO:93 Primer MVT092.

[0768] SEQ ID NO:94 Primer MVT070.

[0769] SEQ ID NO:95 Primer MVT083.

[0770] SEQ ID NO:96 N-terminal amino acid of Cry3Bb polypeptide.

[0771] SEQ ID NO:97 DNA sequence of wild-type cry3Bb gene.

[0772] SEQ ID NO:98 Amino acid sequence of wild-type Cry3Bb polypeptide.

[0773] SEQ ID NO:99 Plantized DNA sequence for cry3Bb.11231 gene.

[0774] SEQ ID NO:100 Amino acid sequence of plantized Cry3Bb.11231polypeptide.

[0775] SEQ ID NO:101 DNA sequence of cry3Bb gene used to prepare SEQ IDNO:99.

[0776] SEQ ID NO:102 DNA sequence of wild-type cry3Bb gene, Genbank#M89794.

[0777] SEQ ID NO:103 DNA sequence of Oligo #1.

[0778] SEQ ID NO:104 DNA sequence of Oligo #2.

[0779] SEQ ID NO:105 DNA sequence of Oligo #3.

[0780] SEQ ID NO:106 DNA sequence of Oligo #4.

[0781] SEQ ID NO:107 DNA sequence of cry3Bb.11098 gene.

[0782] SEQ ID NO:108 Amino acid sequence of Cry3Bb.11098 polypeptide.

[0783] 7.0 References

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0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 113 <210> SEQ ID NO 1<211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400>SEQUENCE: 1 atg aat cca aac aat cga agt gaa cat gat acg ata aag gtt acacct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 15 10 15 aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 2530 cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa gat 192 ThrGlu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gcagtt ggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240 Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 ggagtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288 Gly ValPro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac actata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca 336 Asn Thr IleTrp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gttgaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384 Gln Val GluVal Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gctctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala LeuAla Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aatgcg tta aat tcc tgg aag aaa ttt cac cat tct cgt cgt tct 480 Val Asn AlaLeu Asn Ser Trp Lys Lys Phe His His Ser Arg Arg Ser 145 150 155 160 aaaaga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528 Lys ArgSer Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 catttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His PheArg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctgttt cta cca aca tat gca caa gct gca aat aca cat tta ttg cta 624 Leu PheLeu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 ttaaaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu LysAsp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gatgtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac 720 Asp ValAla Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240act gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768 ThrAsp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 GlySer Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270atg act tta act gta tta gat cta att gta ctt ttc cca ttt tat gat 864 MetThr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285att cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912 IleArg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300ttt acg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca 960 PheThr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315320 act ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330335 tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345350 ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360365 cct agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375380 aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390395 400 tat cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405410 415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420425 430 aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435440 445 cat gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450455 460 gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465470 475 480 tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt acttgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp485 490 495 aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aagatt 1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile500 505 510 act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gcttcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser515 520 525 att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc ctaaaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys530 535 540 gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tcagca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560 gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tctacc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser ThrThr 565 570 575 aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt cttgtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu ValIle 580 585 590 tac att aat aaa act atg aat aaa gat gat gat tta aca tatcaa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr GlnThr 595 600 605 ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggtgat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly AspLys 610 615 620 aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaaaaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu LysIle 625 630 635 640 tat ata gat aag ata gaa ttt atc cca gta caa ttg taa1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQID NO 2 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Recombinant deltaendotoxin <400> SEQUENCE: 2 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn GlnTyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn TyrLys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu AspAsn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val GlyGln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu ThrSer Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp Ala AspPro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp LysLys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu GlnGly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn SerTrp Lys Lys Phe His His Ser Arg Arg Ser 145 150 155 160 Lys Arg Ser GlnAsp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe ArgAsn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu PheLeu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 LeuLys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg ArgGlu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro PheTyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu GlnGlu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile ArgLys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His ThrArg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr TrpSer Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser LysThr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro ValGln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr IleAla Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr LeuGly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys AsnGlu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 HisVal Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu LysIle 500 505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser GlyAla Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu LeuPhe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val ThrLeu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg IleArg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn LysAsp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr AsnSer Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile GlyAla Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp LysIle Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 3 <211> LENGTH:1959 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Recombinant delta endotoxin <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 3 atgaat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48 Met AsnPro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aacagt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96 Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aattca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn SerThr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gacagt tct acg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp SerSer Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg acagga att tct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca tttgct ggg gca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe AlaGly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg ccaagt gat gct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro SerAsp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctgata gat aag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu IleAsp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gagtta cag ggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu LeuGln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aattcc tgg aag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn SerTrp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caagat cga ata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln AspArg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aattcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta ccaaca tat gca caa gct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro ThrTyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gctcaa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala GlnVal Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaattc ctt agt aga caa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu PheLeu Ser Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cattgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr Asp His CysVal Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca acttat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr TyrAsp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act ttaact gta tta gat cta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu ThrVal Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 att cgg ttatac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile Arg Leu TyrSer Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gatcca att ttt tca ctt aat act ctt cag gag tat gga cca 960 Phe Thr Asp ProIle Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 act tttttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe LeuSer Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat ttacag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 Tyr Leu GlnGly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 ggg aaagat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agtata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152 Pro Ser IleGly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tctact gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser ThrGlu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tatcga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr ArgThr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gtatat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val TyrLeu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaaaat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys AsnGlu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 catgta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392 His ValSer Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gatgaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 CysPhe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att 1536 ThrHis Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 ThrGln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 IleIle Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 GluSer Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555560 gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570575 aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585590 tac att aat aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600605 ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615620 aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630635 640 tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 4 <211>LENGTH: 652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <400>SEQUENCE: 4 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val ThrPro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe LeuArg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr ValLys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr GlnSer Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys AlaPhe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu GluTyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln AsnAsn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys ThrPro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp Arg Ile ArgGlu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met ProSer Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr TyrAla Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala GlnVal Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala GluPhe Leu Ser Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr AspHis Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 GlySer Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln ProGly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr SerPro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr AspVal Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr LysVal Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser ThrGln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala GlnAsp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys PheLeu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 ThrHis Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala SerThr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp PheLeu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp LeuThr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser PheVal Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe IlePro Val Gln Leu 645 650 <210> SEQ ID NO 5 <211> LENGTH: 1959 <212> TYPE:DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 5 atg aat cca aac aatcga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caaact aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Gln ThrAsn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaagaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu GluLeu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaagtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gttgta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val ValGly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctcact tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu ThrSer Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gat gct gaccca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp ProTrp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gat aag aaaata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cag ggt cttcaa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu GlnAsn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaaaca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys ThrPro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa gat cga ata agggaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg GluLeu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tcattt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser PheAla Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta cca aca tat gca caagct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gtt ttt ggagaa gaa tgg gga tat tct cca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly GluGlu Trp Gly Tyr Ser Pro Glu 210 215 220 gat gtt gct gaa ttc agt cat agacaa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu Phe Ser His Arg GlnLeu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gtt aat tggtat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gat gca tgggtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp ValLys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gta tta gatcta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp LeuIle Val Leu Phe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggggtt aaa aca gaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gat cca att ttt tcactt aat act ctt cag gag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser LeuAsn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaaaac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu AsnSer Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag ggg att gaattt cat acg cgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aattat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn TyrTrp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agt ata gga tct agtaag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser LysThr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gtacaa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val GlnLys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga act ata gctaat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gttacg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val Tyr Leu Gly Val ThrLys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agtaca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser ThrGln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agt gca caggat tct att gac caa tta ccg cca gaa aca aca 1392 His Val Ser Ala Gln AspSer Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaaaaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu LysAla Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atgcag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cat aga agtgta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His Arg Ser ValAsp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caa ctt ccagta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro ValVal Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggtcca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aattca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn SerIle Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg ttacaa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala Leu Leu GlnArg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cgactt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tac att aataaa act atg aat aaa gat gat gat tta aca tat caa aca 1824 Tyr Ile Asn LysThr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctcgca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu AlaThr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa cttata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tat atagat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 6 <211> LENGTH: 652<212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 6 Met AsnPro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 AsnSer Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 ThrGlu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 AlaVal Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer 145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser GlnAla Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val SerLys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala AsnThr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu GluTrp Gly Tyr Ser Pro Glu 210 215 220 Asp Val Ala Glu Phe Ser His Arg GlnLeu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn TrpTyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp AlaTrp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr ValLeu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu TyrSer Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr AspPro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 ThrPhe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln LysVal 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp ProAsn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser GlnTyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp GlnLeu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr SerHis Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp ArgArg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val AspPhe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro ValVal Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu GlyPro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser SerAsn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 AlaLeu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu LysIle 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 7 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1956) <400> SEQUENCE: 7 atg aat cca aac aat cga agt gaa cat gatacg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caatat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaagaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tctaca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att ttaggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caatca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln SerPhe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct tttatg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe MetAla 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gctaaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaagat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttgcga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caagca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaattc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cattta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tattct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 gat gtt gct gaa ttc tat cgt aga caa tta aaa ctt acacaa caa tac 720 Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr GlnGln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga ttaaat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu AsnGly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgtttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttccca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa ctaaca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile 290 295 300 ttt acg gat cca att ttt tca ctt aat act ctt caggag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln GluTyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaacct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt cttcaa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu GlnPro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aattat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcccca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc tttgat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcggct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat tttagt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gattca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caatta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt catcag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt ggaaca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly ThrIle Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aataca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tatgcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca ggagga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa tttaaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gtaaga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aattca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaagat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tctaat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaatct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa tttatc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 8 <211> LENGTH: 652 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <400> SEQUENCE: 8 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu GlnThr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser SerThr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro PheAla Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val GluVal Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys AlaLeu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 ValAsn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu LeuLeu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu ThrGln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys PheAsn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu IleVal Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe SerLeu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln GlyIle Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn AsnGly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro GluThr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala TyrAla Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile AlaLys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln ArgTyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile AsnLys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 AsnGlu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ IDNO 9 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Recombinant deltaendotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956)<400> SEQUENCE: 9 atg aat cca aac aat cga agt gaa cat gat acg ata aaggtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys ValThr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caa tat cct ttagct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt ttaaga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu ArgMet 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaagat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 6570 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaaagt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaagtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta ttgcta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tcagaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu210 215 220 gat gtt gct gaa ttc tat aat aga caa tta aaa ctt aca caa caatac 720 Asp Val Ala Glu Phe Tyr Asn Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 tct gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggttta aga 768 Ser Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly LeuArg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgcaga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg ArgGlu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttc cca ttttat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe TyrAsp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca agagac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 ttt acg gat cca att ttt tca ctt aat act ctt cag gag tatgga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr GlyPro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaa cct cattta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt ctt caa cctggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro GlyTyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gtagaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val GluThr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcc cca ttttat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc ttt gat ggacaa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly GlnLys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcg gct tggccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp ProAsn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caatat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gat tca aaaaga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys ArgAsn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caa tta ccgcca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro ProGlu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt cat cag cttaat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt gga aca attcca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile ProPhe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aat aca attgat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile AspAla Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tat gcc ttgtct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca gga gga aattta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa ttt aaa gttaca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val ThrLeu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gta aga atacgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aat tca aacaat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaa gat gatgat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp AspLeu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tct aat atgggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaa tct ttcgtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe ValSer Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa ttt atc ccagta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 10 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <400> SEQUENCE: 10 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn HisAsn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu LeuAsn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser ValVal Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser AspAla Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu IleAsp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala GluLeu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys ArgSer Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 HisPhe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210215 220 Asp Val Ala Glu Phe Tyr Asn Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 Ser Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn GlyLeu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu PhePro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn ThrLeu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn SerIle Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe AsnTyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr GluPro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr ArgThr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 ValTyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe PheThr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp AlaGlu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly AsnLeu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg ValArg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr MetAsn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala ThrThr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 11 <211>LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE:11 atg aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 actgaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gttggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val GlyThr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gttcca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val ProPhe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act atatgg cca agt gat gct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaagta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu ValLeu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct cttgca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu AlaGlu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcgtta aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa agaagc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg SerGln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat tttcgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe ArgAsn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg tttcta cca aca tat gca caa gct gca aat aca cat tta ttg cta 624 Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaagat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gttgct gaa ttc tat acc aga caa tta aaa ctt aca caa caa tac 720 Asp Val AlaGlu Phe Tyr Thr Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 actgac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr AspHis Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggttca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly SerThr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atgact tta act gta tta gat cta att gta ctt ttc cca ttt tat gat 864 Met ThrLeu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 attcgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 tttacg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca 960 Phe ThrAsp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008 ThrPhe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 GlyLys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152 ProSer Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 tat cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425430 aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 cat gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455460 gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470475 480 tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485490 495 aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515520 525 att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530535 540 gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545550 555 560 gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct accact 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr565 570 575 aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtcatc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 tac att aat aaa act atg aat aaa gat gat gat tta aca tat caaaca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr595 600 605 ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gataag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys610 615 620 aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaaatc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO12 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin<400> SEQUENCE: 12 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile LysVal Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn SerThr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser PheTyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro TrpLys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly LeuGln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp ArgIle Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp ValAla Glu Phe Tyr Thr Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu TyrGly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg LeuGln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr IleThr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln LysLeu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly ValThr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val SerAla Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe LeuLys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu AsnSer Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg TyrAla Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp AspAsp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala GluSer Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 13 <211> LENGTH: 1959<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Recombinant delta endotoxin <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 13 atg aat ccaaac aat cga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro AsnAsn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaattg caa act aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu LeuGln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca acacta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tctacg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser ThrGlu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga atttct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile SerVal Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggggca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gatgct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp AlaAsp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gataag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp LysLys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cagggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tggaag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa gat cgaata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg IleArg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atgccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met ProSer Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta cca aca tatgca caa gct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr AlaGln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gttttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val PheGly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tatcat aga caa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gttaat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val AsnTrp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gatgca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp AlaTrp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gtatta gat cta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 att aat tta tac tcaaaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile Asn Leu Tyr Ser LysGly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gat cca attttt tca ctt aat act ctt cag gag tat gga cca 960 Phe Thr Asp Pro Ile PheSer Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agtata gaa aac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag gggatt gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly IleGlu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tctttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser PheAsn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agt ata ggatct agt aag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaacct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga actata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr IleAla Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gta tat ttaggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaaact agt aca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agtgca cag gat tct att gac caa tta ccg cca gaa aca aca 1392 His Val Ser AlaGln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa ccactt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttctta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe LeuMet Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cataga agt gta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His ArgSer Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caactt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att attgaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile GluGly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tctagt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser SerAsn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gccttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aactta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776 Asn LeuArg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tacatt aat aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824 Tyr IleAsn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 tttgat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aatgaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn GluLeu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp LysIle Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 14 <211> LENGTH:652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 14Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 2530 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 4045 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 5560 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 7075 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe GluAsp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu PheSer Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe AlaVal Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe GlyGlu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys ValAsn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr TyrAsp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile AsnLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 PheThr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu ThrArg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp GlyGln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp PheSer Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser IleAsp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys AlaTyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg SerVal Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile IleGlu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 GluSer Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr GlnThr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser GlyAsp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser AsnGlu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 15 <211> LENGTH: 1959 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1956) <400> SEQUENCE: 15 atg aat cca aac aat cga agt gaacat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cataat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His AsnGln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aattat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn TyrLys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gacaac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cagatt tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttttat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe TyrGln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aaggct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys AlaPhe Met Ala 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gagtat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu TyrAla Lys Ser 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aatttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn PheGlu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct ttaagt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttttct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe SerGln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtttcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val SerLys Phe Glu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aataca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgggga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp GlyTyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaactt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys LeuThr Gln Gln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gttgga tta aat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa tttaac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe AsnArg Phe Arg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gtactt ttc cca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val LeuPhe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa acagaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 ttt acg gat cca att ttt tta ctt act acgctt cag aag tac gga cca 960 Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr LeuGln Lys Tyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct attcga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile ArgLys Pro His Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acgcgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tctggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca attact tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile ThrSer Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag ctaagc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gacgta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp ValAla Ala Trp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gttgat ttt agt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val AspPhe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa acatat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct attgac caa tta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile AspGln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tatagt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr SerHis Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgtcgt gga aca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac tttttt aat aca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe PheAsn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaagca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys AlaTyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttcaca gga gga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gctaaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala LysPhe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tatcgt gta aga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr ArgVal Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtgcaa aat tca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atgaat aaa gat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act actaat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr AsnSer Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata ggagca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag atagaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile ProVal Gln Leu 645 650 <210> SEQ ID NO 16 <211> LENGTH: 652 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 16 Met Asn ProAsn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 GlyVal Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 AsnThr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser LysPhe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu TrpGly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln LeuLys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala TrpVal Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr SerLys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp ProIle Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly Pro 305 310 315 320 Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro AsnGly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser LysArg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser HisGln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp PhePhe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val ValLys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser AsnSer Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 AsnLeu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650<210> SEQ ID NO 17 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1956) <400> SEQUENCE: 17 atg aat cca aac aat cga agt gaa cat gatacg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caatat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaagaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tctaca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att ttaggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caatca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln SerPhe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct tttatg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe MetAla 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gctaaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaagat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttgcga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caagca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaattc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cattta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tattct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt acacaa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr GlnGln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga ttaaat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu AsnGly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgtttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttccca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa ctaaca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile 290 295 300 ttt acg gat cca att ttt acc ctt aat aca cta cagaag tgc gga cca 960 Phe Thr Asp Pro Ile Phe Thr Leu Asn Thr Leu Gln LysCys Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaacct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt cttcaa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu GlnPro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aattat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcccca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc tttgat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcggct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat tttagt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gattca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caatta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt catcag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt ggaaca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly ThrIle Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aataca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tatgcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca ggagga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa tttaaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gtaaga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aattca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaagat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tctaat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaatct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa tttatc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 18 <211> LENGTH: 652 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <400> SEQUENCE: 18 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu GlnThr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser SerThr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro PheAla Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val GluVal Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys AlaLeu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 ValAsn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu LeuLeu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu ThrGln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys PheAsn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu IleVal Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe ThrLeu Asn Thr Leu Gln Lys Cys Gly Pro 305 310 315 320 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln GlyIle Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn AsnGly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro GluThr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala TyrAla Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile AlaLys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln ArgTyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile AsnLys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 AsnGlu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ IDNO 19 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Recombinant deltaendotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956)<400> SEQUENCE: 19 atg aat cca aac aat cga agt gaa cat gat acg ata aaggtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys ValThr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caa tat cct ttagct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt ttaaga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu ArgMet 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaagat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 6570 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaaagt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaagtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta ttgcta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tcagaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caatac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggttta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly LeuArg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgcaga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg ArgGlu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttc cca ttttat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe TyrAsp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca agagac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 ttt acg gat cca att ttt gcc gtt aat act ctg tgg gaa tacgga cca 960 Phe Thr Asp Pro Ile Phe Ala Val Asn Thr Leu Trp Glu Tyr GlyPro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaa cct cattta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt ctt caa cctggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro GlyTyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gtagaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val GluThr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcc cca ttttat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc ttt gat ggacaa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly GlnLys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcg gct tggccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp ProAsn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caatat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gat tca aaaaga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys ArgAsn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caa tta ccgcca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro ProGlu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt cat cag cttaat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt gga aca attcca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile ProPhe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aat aca attgat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile AspAla Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tat gcc ttgtct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca gga gga aattta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa ttt aaa gttaca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val ThrLeu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gta aga atacgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aat tca aacaat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaa gat gatgat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp AspLeu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tct aat atgggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaa tct ttcgtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe ValSer Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa ttt atc ccagta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 20 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <400> SEQUENCE: 20 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn HisAsn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu LeuAsn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser ValVal Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser AspAla Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu IleAsp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala GluLeu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys ArgSer Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 HisPhe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn GlyLeu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu PhePro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe Ala Val Asn ThrLeu Trp Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn SerIle Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe AsnTyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr GluPro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr ArgThr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 ValTyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe PheThr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp AlaGlu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly AsnLeu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg ValArg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr MetAsn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala ThrThr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 21 <211>LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE:21 atg aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 actgaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gttggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val GlyThr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gttcca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val ProPhe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act atatgg cca agt gat gct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaagta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu ValLeu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct cttgca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu AlaGlu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcgtta aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa agaagc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg SerGln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat tttcgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe ArgAsn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg tttcta cca aca tat gca caa gct gca aat aca cat tta ttg cta 624 Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaagat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gttgct gaa ttc tat cgt aga caa tta aaa ctt aca caa caa tac 720 Asp Val AlaGlu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 actgac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr AspHis Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggttca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly SerThr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atgact tta act gta tta gat cta att gta ctt ttc cca ttt tat gat 864 Met ThrLeu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 attcgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 tttacg gat cca att ttt tta ctt act acg ctt cag aag tac gga cca 960 Phe ThrAsp Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly Pro 305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008 ThrPhe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 GlyLys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152 ProSer Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 tat cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425430 aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 cat gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455460 gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470475 480 tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485490 495 aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515520 525 att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530535 540 gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545550 555 560 gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct accact 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr565 570 575 aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtcatc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 tac att aat aaa act atg aat aaa gat gat gat tta aca tat caaaca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr595 600 605 ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gataag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys610 615 620 aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaaatc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO22 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin<400> SEQUENCE: 22 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile LysVal Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn SerThr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser PheTyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro TrpLys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly LeuGln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp ArgIle Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp ValAla Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys TyrGly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg LeuGln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr IleThr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln LysLeu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly ValThr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val SerAla Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe LeuLys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu AsnSer Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg TyrAla Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp AspAsp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala GluSer Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 23 <211> LENGTH: 1959<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Recombinant delta endotoxin <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 23 atg aat ccaaac aat cga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro AsnAsn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaattg caa act aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu LeuGln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca acacta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tctacg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser ThrGlu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga atttct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile SerVal Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggggca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gatgct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp AlaAsp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gataag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp LysLys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cagggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tggaag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa gat cgaata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg IleArg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atgccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met ProSer Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta cca aca tatgca caa gct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr AlaGln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gttttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val PheGly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tatcat aga caa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gttaat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val AsnTrp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gatgca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp AlaTrp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gtatta gat cta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tcaaaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser LysGly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gat cca attttt acg cca acc acc cta cag gat tac gga cca 960 Phe Thr Asp Pro Ile PheThr Pro Thr Thr Leu Gln Asp Tyr Gly Pro 305 310 315 320 act ttt ttg agtata gaa aac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag gggatt gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly IleGlu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tctttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser PheAsn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agt ata ggatct agt aag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaacct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga actata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr IleAla Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gta tat ttaggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaaact agt aca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agtgca cag gat tct att gac caa tta ccg cca gaa aca aca 1392 His Val Ser AlaGln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa ccactt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttctta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe LeuMet Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cataga agt gta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His ArgSer Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caactt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att attgaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile GluGly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tctagt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser SerAsn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gccttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aactta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776 Asn LeuArg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tacatt aat aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824 Tyr IleAsn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 tttgat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aatgaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn GluLeu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp LysIle Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 24 <211> LENGTH:652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 24Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 2530 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 4045 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 5560 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 7075 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe GluAsp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu PheSer Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe AlaVal Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe GlyGlu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys ValAsn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr TyrAsp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 PheThr Asp Pro Ile Phe Thr Pro Thr Thr Leu Gln Asp Tyr Gly Pro 305 310 315320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu ThrArg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp GlyGln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp PheSer Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser IleAsp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys AlaTyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg SerVal Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile IleGlu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 GluSer Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr GlnThr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser GlyAsp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser AsnGlu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 25 <211> LENGTH: 1959 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1956) <400> SEQUENCE: 25 atg aat cca aac aat cga agt gaacat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cataat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His AsnGln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aattat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn TyrLys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gacaac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cagatt tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttttat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe TyrGln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aaggct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys AlaPhe Met Ala 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gagtat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu TyrAla Lys Ser 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aatttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn PheGlu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct ttaagt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttttct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe SerGln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtttcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val SerLys Phe Glu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aataca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgggga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp GlyTyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaactt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys LeuThr Gln Gln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gttgga tta aat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa tttaac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe AsnArg Phe Arg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gtactt ttc cca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val LeuPhe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa acagaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 ttt acg gat cca att ttt gcc ctg aat acctta gac gag tac gga cca 960 Phe Thr Asp Pro Ile Phe Ala Leu Asn Thr LeuAsp Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct attcga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile ArgLys Pro His Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acgcgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tctggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca attact tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile ThrSer Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag ctaagc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gacgta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp ValAla Ala Trp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gttgat ttt agt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val AspPhe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa acatat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct attgac caa tta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile AspGln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tatagt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr SerHis Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgtcgt gga aca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac tttttt aat aca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe PheAsn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaagca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys AlaTyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttcaca gga gga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gctaaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala LysPhe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tatcgt gta aga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr ArgVal Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtgcaa aat tca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atgaat aaa gat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act actaat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr AsnSer Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata ggagca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag atagaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile ProVal Gln Leu 645 650 <210> SEQ ID NO 26 <211> LENGTH: 652 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 26 Met Asn ProAsn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 GlyVal Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 AsnThr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser LysPhe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu TrpGly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln LeuLys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala TrpVal Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr SerLys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp ProIle Phe Ala Leu Asn Thr Leu Asp Glu Tyr Gly Pro 305 310 315 320 Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro AsnGly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser LysArg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser HisGln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp PhePhe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val ValLys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser AsnSer Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 AsnLeu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650<210> SEQ ID NO 27 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1956) <400> SEQUENCE: 27 atg aat cca aac aat cga agt gaa cat gatacg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caatat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaagaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tctaca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att ttaggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caatca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln SerPhe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct tttatg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe MetAla 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gctaaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaagat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttgcga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caagca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaattc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cattta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tattct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt acacaa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr GlnGln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga ttaaat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu AsnGly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgtttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttccca ttt tac gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp 275 280 285 act agg cga ttc aga aag ggg gtt aaa aca gaa ctaaca aga gac att 912 Thr Arg Arg Phe Arg Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile 290 295 300 ttt acg gat cca att ttt tca ctt aat act ctt caggag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln GluTyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaacct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt cttcaa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu GlnPro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aattat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcccca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc tttgat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcggct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat tttagt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gattca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caatta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt catcag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt ggaaca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly ThrIle Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aataca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tatgcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca ggagga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa tttaaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gtaaga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aattca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaagat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tctaat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaatct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa tttatc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 28 <211> LENGTH: 652 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <400> SEQUENCE: 28 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu GlnThr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser SerThr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro PheAla Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val GluVal Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys AlaLeu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 ValAsn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu LeuLeu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu ThrGln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys PheAsn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu IleVal Leu Phe Pro Phe Tyr Asp 275 280 285 Thr Arg Arg Phe Arg Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe SerLeu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln GlyIle Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn AsnGly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro GluThr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala TyrAla Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile AlaLys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln ArgTyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile AsnLys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 AsnGlu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ IDNO 29 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Recombinant deltaendotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956)<400> SEQUENCE: 29 atg aat cca aac aat cga agt gaa cat gat acg ata aaggtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys ValThr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caa tat cct ttagct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt ttaaga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu ArgMet 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaagat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 6570 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaaagt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaagtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta ttgcta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tcagaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu210 215 220 gat gtt gct gaa ttc tat cgt aga caa tta aaa ctt aca caa caatac 720 Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggttta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly LeuArg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgcaga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg ArgGlu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttc cca ttttat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe TyrAsp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca agagac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 ttt acg gat cca att ttt tta ctt aat act ctt cag gag tatgga cca 960 Phe Thr Asp Pro Ile Phe Leu Leu Asn Thr Leu Gln Glu Tyr GlyPro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaa cct cattta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt ctt caa cctggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro GlyTyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gtagaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val GluThr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcc cca ttttat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc ttt gat ggacaa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly GlnLys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcg gct tggccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp ProAsn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caatat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gat tca aaaaga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys ArgAsn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caa tta ccgcca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro ProGlu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt cat cag cttaat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt gga aca attcca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile ProPhe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aat aca attgat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile AspAla Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tat gcc ttgtct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca gga gga aattta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa ttt aaa gttaca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val ThrLeu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gta aga atacgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aat tca aacaat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaa gat gatgat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp AspLeu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tct aat atgggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaa tct ttcgtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe ValSer Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa ttt atc ccagta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 30 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <400> SEQUENCE: 30 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn HisAsn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu LeuAsn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser ValVal Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser AspAla Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu IleAsp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala GluLeu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys ArgSer Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 HisPhe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210215 220 Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn GlyLeu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu PhePro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe Leu Leu Asn ThrLeu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn SerIle Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe AsnTyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr GluPro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr ArgThr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 ValTyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe PheThr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp AlaGlu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly AsnLeu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg ValArg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr MetAsn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala ThrThr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 31 <211>LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE:31 atg aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 actgaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gttggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val GlyThr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gttcca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val ProPhe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act atatgg cca agt gat gct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaagta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu ValLeu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct cttgca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu AlaGlu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcgtta aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa agaagc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg SerGln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat tttcgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe ArgAsn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg tttcta cca aca tat gca caa gct gca aat aca cat tta ttg cta 624 Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaagat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gttgct gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac 720 Asp Val AlaGlu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 actgac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr AspHis Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggttca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly SerThr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atgact tta act gta tta gat cta att gta ctt ttc cca ttt tat gat 864 Met ThrLeu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 attcgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 tttacg gat cca att ttt atc ctc aat acg cta cag gag tac gga cca 960 Phe ThrAsp Pro Ile Phe Ile Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008 ThrPhe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 GlyLys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152 ProSer Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 tat cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425430 aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 cat gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455460 gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470475 480 tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485490 495 aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515520 525 att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530535 540 gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545550 555 560 gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct accact 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr565 570 575 aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtcatc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 tac att aat aaa act atg aat aaa gat gat gat tta aca tat caaaca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr595 600 605 ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gataag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys610 615 620 aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaaatc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO32 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin<400> SEQUENCE: 32 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile LysVal Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn SerThr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser PheTyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro TrpLys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly LeuGln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp ArgIle Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp ValAla Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 Phe Thr Asp Pro Ile Phe Ile Leu Asn Thr Leu Gln Glu TyrGly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg LeuGln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr IleThr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln LysLeu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly ValThr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val SerAla Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe LeuLys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu AsnSer Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg TyrAla Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp AspAsp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala GluSer Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 33 <211> LENGTH: 1959<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Recombinant delta endotoxin <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 33 atg aat ccaaac aat cga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro AsnAsn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaattg caa act aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu LeuGln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca acacta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tctacg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser ThrGlu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga atttct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile SerVal Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggggca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gatgct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp AlaAsp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gataag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp LysLys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cagggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tggaag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa gat cgaata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg IleArg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atgccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met ProSer Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta cca aca tatgca caa gct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr AlaGln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gttttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val PheGly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tatcat aga caa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gttaat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val AsnTrp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gatgca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp AlaTrp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gtatta gat cta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tcaaaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser LysGly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gat cca attttt atc cta cat acg ctg cag gag tac gga cca 960 Phe Thr Asp Pro Ile PheIle Leu His Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agtata gaa aac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag gggatt gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly IleGlu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tctttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser PheAsn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agt ata ggatct agt aag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaacct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga actata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr IleAla Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gta tat ttaggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaaact agt aca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agtgca cag gat tct att gac caa tta ccg cca gaa aca aca 1392 His Val Ser AlaGln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa ccactt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttctta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe LeuMet Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cataga agt gta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His ArgSer Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caactt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att attgaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile GluGly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tctagt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser SerAsn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gccttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aactta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776 Asn LeuArg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tacatt aat aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824 Tyr IleAsn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 tttgat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aatgaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn GluLeu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp LysIle Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 34 <211> LENGTH:652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 34Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 2530 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 4045 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 5560 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 7075 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe GluAsp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu PheSer Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe AlaVal Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe GlyGlu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys ValAsn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr TyrAsp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 PheThr Asp Pro Ile Phe Ile Leu His Thr Leu Gln Glu Tyr Gly Pro 305 310 315320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu ThrArg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp GlyGln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp PheSer Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser IleAsp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys AlaTyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg SerVal Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile IleGlu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 GluSer Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr GlnThr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser GlyAsp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser AsnGlu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 35 <211> LENGTH: 1959 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1956) <400> SEQUENCE: 35 atg aat cca aac aat cga agt gaacat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cataat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His AsnGln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aattat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn TyrLys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gacaac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cagatt tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttttat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe TyrGln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aaggct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys AlaPhe Met Ala 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gagtat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu TyrAla Lys Ser 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aatttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn PheGlu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct ttaagt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttttct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe SerGln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtttcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val SerLys Phe Glu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aataca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgggga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp GlyTyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaactt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys LeuThr Gln Gln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gttgga tta aat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa tttaac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe AsnArg Phe Arg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gtactt ttc cca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val LeuPhe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa acagaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 ttt acg gat cca att ttt tcc ctc gtt aaccta atg gtg tac gga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Val Asn LeuMet Val Tyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct attcga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile ArgLys Pro His Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acgcgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tctggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca attact tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile ThrSer Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag ctaagc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gacgta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp ValAla Ala Trp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gttgat ttt agt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val AspPhe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa acatat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct attgac caa tta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile AspGln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tatagt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr SerHis Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgtcgt gga aca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac tttttt aat aca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe PheAsn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaagca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys AlaTyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttcaca gga gga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gctaaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala LysPhe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tatcgt gta aga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr ArgVal Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtgcaa aat tca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atgaat aaa gat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act actaat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr AsnSer Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata ggagca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag atagaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile ProVal Gln Leu 645 650 <210> SEQ ID NO 36 <211> LENGTH: 652 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 36 Met Asn ProAsn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 GlyVal Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 AsnThr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser LysPhe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu TrpGly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln LeuLys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala TrpVal Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr SerLys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp ProIle Phe Ser Leu Val Asn Leu Met Val Tyr Gly Pro 305 310 315 320 Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro AsnGly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser LysArg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser HisGln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp PhePhe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val ValLys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser AsnSer Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 AsnLeu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650<210> SEQ ID NO 37 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1956) <400> SEQUENCE: 37 atg aat cca aac aat cga agt gaa cat gatacg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caatat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaagaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tctaca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att ttaggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caatca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln SerPhe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct tttatg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe MetAla 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gctaaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaagat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttgcga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caagca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaattc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cattta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tattct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt acacaa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr GlnGln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga ttaaat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu AsnGly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgtttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttccca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa ctaaca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile 290 295 300 ttt acg gat cca att ttt tct ctt agg aca cca cttgcg tac gga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Arg Thr Pro Leu AlaTyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaacct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt cttcaa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu GlnPro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aattat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcccca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc tttgat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcggct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat tttagt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gattca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caatta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt catcag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt ggaaca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly ThrIle Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aataca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tatgcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca ggagga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa tttaaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gtaaga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aattca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaagat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tctaat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaatct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa tttatc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 38 <211> LENGTH: 652 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <400> SEQUENCE: 38 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu GlnThr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser SerThr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro PheAla Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val GluVal Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys AlaLeu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 ValAsn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu LeuLeu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu ThrGln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys PheAsn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu IleVal Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe SerLeu Arg Thr Pro Leu Ala Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln GlyIle Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn AsnGly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro GluThr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala TyrAla Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile AlaLys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln ArgTyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile AsnLys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 AsnGlu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ IDNO 39 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Recombinant deltaendotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956)<400> SEQUENCE: 39 atg aat cca aac aat cga agt gaa cat gat acg ata aaggtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys ValThr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caa tat cct ttagct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt ttaaga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu ArgMet 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaagat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 6570 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaaagt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaagtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta ttgcta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tcagaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caatac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggttta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly LeuArg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgcaga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg ArgGlu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttc cca tttttc aat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe PheAsn 275 280 285 att ttg ctt tac agt aaa ggg gtt aaa aca gaa cta aca agagac att 912 Ile Leu Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 ttt acg gat cca att ttt tca ctt aat act ctt cag gag tatgga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr GlyPro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaa cct cattta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt ctt caa cctggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro GlyTyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gtagaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val GluThr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcc cca ttttat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc ttt gat ggacaa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly GlnLys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcg gct tggccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp ProAsn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caatat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gat tca aaaaga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys ArgAsn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caa tta ccgcca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro ProGlu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt cat cag cttaat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt gga aca attcca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile ProPhe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aat aca attgat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile AspAla Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tat gcc ttgtct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca gga gga aattta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa ttt aaa gttaca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val ThrLeu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gta aga atacgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aat tca aacaat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaa gat gatgat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp AspLeu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tct aat atgggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaa tct ttcgtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe ValSer Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa ttt atc ccagta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 40 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <400> SEQUENCE: 40 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn HisAsn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu LeuAsn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser ValVal Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser AspAla Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu IleAsp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala GluLeu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys ArgSer Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 HisPhe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn GlyLeu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu PhePro Phe Phe Asn 275 280 285 Ile Leu Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn ThrLeu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn SerIle Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe AsnTyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr GluPro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr ArgThr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 ValTyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe PheThr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp AlaGlu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly AsnLeu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg ValArg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr MetAsn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala ThrThr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 41 <211>LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE:41 atg aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 actgaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gttggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val GlyThr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gttcca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val ProPhe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act atatgg cca agt gat gct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaagta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu ValLeu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct cttgca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu AlaGlu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcgtta aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa agaagc caa gat cga ata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg SerGln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat tttcgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe ArgAsn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg tttcta cca aca tat gca caa gct gca aat aca cat tta ttg cta 624 Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaagat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gttgct gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac 720 Asp Val AlaGlu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 actgac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr AspHis Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggttca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly SerThr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atgact tta act gta tta gat cta att gta ctt ttc cca ttt tat gat 864 Met ThrLeu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 attgtg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile ValLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 tttacg gat cca att ttt tca ctt aat act ctt cag gag tat gga cca 960 Phe ThrAsp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008 ThrPhe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 GlyLys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152 ProSer Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 tat cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425430 aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 cat gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455460 gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470475 480 tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485490 495 aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515520 525 att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530535 540 gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545550 555 560 gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct accact 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr565 570 575 aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtcatc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 tac att aat aaa act atg aat aaa gat gat gat tta aca tat caaaca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr595 600 605 ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gataag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys610 615 620 aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaaatc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO42 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin<400> SEQUENCE: 42 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile LysVal Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn SerThr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser PheTyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro TrpLys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly LeuGln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp ArgIle Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp ValAla Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp275 280 285 Ile Val Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu TyrGly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg LeuGln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr IleThr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln LysLeu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly ValThr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val SerAla Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe LeuLys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu AsnSer Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg TyrAla Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp AspAsp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala GluSer Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 43 <211> LENGTH: 1959<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Recombinant delta endotoxin <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 43 atg aat ccaaac aat cga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro AsnAsn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaattg caa act aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu LeuGln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca acacta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tctacg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser ThrGlu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga atttct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile SerVal Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggggca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gatgct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp AlaAsp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gataag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp LysLys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cagggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tggaag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa ggt cgaata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Gly Arg IleArg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atgccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met ProSer Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta cca aca tatgca caa gct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr AlaGln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gttttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val PheGly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tatcat aga caa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gttaat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val AsnTrp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gatgca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp AlaTrp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gtatta gat cta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tcaaaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser LysGly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gat cca attttt tca ctt aat act ctt cag gag tat gga cca 960 Phe Thr Asp Pro Ile PheSer Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agtata gaa aac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag gggatt gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly IleGlu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tctttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser PheAsn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agt ata ggatct agt aag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaacct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga actata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr IleAla Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gta tat ttaggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaaact agt aca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agtgca cag gat tct att gac caa tta ccg cca gaa aca aca 1392 His Val Ser AlaGln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa ccactt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttctta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe LeuMet Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cataga agt gta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His ArgSer Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caactt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att attgaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile GluGly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tctagt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser SerAsn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gccttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aactta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776 Asn LeuArg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tacatt aat aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824 Tyr IleAsn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 tttgat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aatgaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn GluLeu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp LysIle Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 44 <211> LENGTH:652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 44Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 2530 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 4045 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 5560 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 7075 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe GluAsp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 Lys Arg Ser Gln Gly Arg Ile Arg Glu Leu PheSer Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe AlaVal Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe GlyGlu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys ValAsn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr TyrAsp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 PheThr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu ThrArg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp GlyGln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp PheSer Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser IleAsp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys AlaTyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg SerVal Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile IleGlu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 GluSer Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr GlnThr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser GlyAsp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser AsnGlu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 45 <211> LENGTH: 1959 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1956) <400> SEQUENCE: 45 atg aat cca aac aat cga agt gaacat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cataat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His AsnGln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aattat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn TyrLys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gacaac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cagatt tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttttat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe TyrGln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aaggct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys AlaPhe Met Ala 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gagtat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu TyrAla Lys Ser 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aatttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn PheGlu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct ttaagt ttg cga aat 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Asn 145 150 155 160 cca cac agc caa ggt cga ata agg gaa ctt ttttct caa gca gaa agt 528 Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe SerGln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtttcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val SerLys Phe Glu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aataca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgggga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp GlyTyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaactt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys LeuThr Gln Gln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gttgga tta aat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa tttaac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe AsnArg Phe Arg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gtactt ttc cca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val LeuPhe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa acagaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 ttt acg gat cca att ttt tca ctt aat actctt cag gag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr LeuGln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct attcga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile ArgLys Pro His Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acgcgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tctggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca attact tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile ThrSer Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag ctaagc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gacgta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp ValAla Ala Trp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gttgat ttt agt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val AspPhe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa acatat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct attgac caa tta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile AspGln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tatagt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr SerHis Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgtcgt gga aca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac tttttt aat aca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe PheAsn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaagca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys AlaTyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttcaca gga gga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gctaaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala LysPhe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tatcgt gta aga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr ArgVal Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtgcaa aat tca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atgaat aaa gat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act actaat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr AsnSer Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata ggagca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag atagaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile ProVal Gln Leu 645 650 <210> SEQ ID NO 46 <211> LENGTH: 652 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 46 Met Asn ProAsn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 GlyVal Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 AsnThr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Asn145 150 155 160 Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser LysPhe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu TrpGly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln LeuLys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala TrpVal Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr SerLys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp ProIle Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro AsnGly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser LysArg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser HisGln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp PhePhe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val ValLys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser AsnSer Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 AsnLeu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650<210> SEQ ID NO 47 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1956) <400> SEQUENCE: 47 atg aat cca aac aat cga agt gaa cat gatacg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caatat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaagaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tctaca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att ttaggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caatca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln SerPhe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct tttatg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe MetAla 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gctaaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaagat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttgcga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caagca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaattc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cattta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tattct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt acacaa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr GlnGln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga ttaaat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu AsnGly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgtttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttccca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp 275 280 285 gtt cgg tta tac cca aaa ggg gtt aaa aca gaa ctaaca aga gac att 912 Val Arg Leu Tyr Pro Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile 290 295 300 ttt acg gat cca att ttt tca ctt aat act ctt caggag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln GluTyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaacct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt cttcaa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu GlnPro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aattat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcccca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc tttgat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcggct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat tttagt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gattca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caatta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt catcag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt ggaaca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly ThrIle Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aataca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tatgcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca ggagga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa tttaaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gtaaga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aattca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaagat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tctaat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaatct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa tttatc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 48 <211> LENGTH: 652 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <400> SEQUENCE: 48 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu GlnThr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser SerThr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro PheAla Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val GluVal Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys AlaLeu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 ValAsn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu LeuLeu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu ThrGln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys PheAsn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu IleVal Leu Phe Pro Phe Tyr Asp 275 280 285 Val Arg Leu Tyr Pro Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe SerLeu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln GlyIle Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn AsnGly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro GluThr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala TyrAla Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile AlaLys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln ArgTyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile AsnLys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 AsnGlu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ IDNO 49 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Recombinant deltaendotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956)<400> SEQUENCE: 49 atg aat cca aac aat cga agt gaa cat gat acg ata aaggtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys ValThr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caa tat cct ttagct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt ttaaga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu ArgMet 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaagat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 6570 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga aat480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Asn 145150 155 160 cca cac agc caa ggt cga ata agg gaa ctt ttt tct caa gca gaaagt 528 Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaagtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta ttgcta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tcagaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caatac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggttta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly LeuArg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgcaga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg ArgGlu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttc cca ttttat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe TyrAsp 275 280 285 gtt cgg tta tac cca aaa ggg gtt aaa aca gaa cta aca agagac att 912 Val Arg Leu Tyr Pro Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 ttt acg gat cca att ttt tca ctt aat act ctt cag gag tatgga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr GlyPro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaa cct cattta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt ctt caa cctggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro GlyTyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gtagaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val GluThr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcc cca ttttat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc ttt gat ggacaa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly GlnLys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcg gct tggccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp ProAsn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caatat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gat tca aaaaga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys ArgAsn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caa tta ccgcca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro ProGlu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt cat cag cttaat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt gga aca attcca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile ProPhe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aat aca attgat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile AspAla Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tat gcc ttgtct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca gga gga aattta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa ttt aaa gttaca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val ThrLeu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gta aga atacgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aat tca aacaat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaa gat gatgat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp AspLeu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tct aat atgggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaa tct ttcgtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe ValSer Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa ttt atc ccagta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 50 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <400> SEQUENCE: 50 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn HisAsn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu LeuAsn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser ValVal Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser AspAla Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu IleAsp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala GluLeu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Asn 145 150 155 160 Pro HisSer Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 HisPhe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn GlyLeu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu PhePro Phe Tyr Asp 275 280 285 Val Arg Leu Tyr Pro Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn ThrLeu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn SerIle Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe AsnTyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr GluPro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr ArgThr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 ValTyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe PheThr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp AlaGlu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly AsnLeu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg ValArg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr MetAsn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala ThrThr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 51 <211>LENGTH: 1956 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1953) <400> SEQUENCE:51 atg aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 actgaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gttggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val GlyThr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gttcca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val ProPhe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act atatgg cca agt gaa gac cca tgg aag gct ttt atg gca caa 336 Asn Thr Ile TrpPro Ser Glu Asp Pro Trp Lys Ala Phe Met Ala Gln 100 105 110 gtt gaa gtactg ata gat aag aaa ata gag gag tat gct aaa agt aaa 384 Val Glu Val LeuIle Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys 115 120 125 gct ctt gcagag tta cag ggt ctt caa aat aat ttc gaa gat tat gtt 432 Ala Leu Ala GluLeu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val 130 135 140 aat gcg ttaaat tcc tgg aag aaa aca cct tta agt ttg cga agt aaa 480 Asn Ala Leu AsnSer Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser Lys 145 150 155 160 aga agccaa gat cga ata agg gaa ctt ttt tct caa gca gaa agt cat 528 Arg Ser GlnAsp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His 165 170 175 ttt cgtaat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg ctg 576 Phe Arg AsnSer Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val Leu 180 185 190 ttt ctacca aca tat gca caa gct gca aat aca cat tta ttg cta tta 624 Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu Leu 195 200 205 aaa gatgct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa gat 672 Lys Asp AlaGln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp 210 215 220 gtt gctgaa ttt tat cat aga caa tta aaa ctt aca caa caa tac act 720 Val Ala GluPhe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr 225 230 235 240 gaccat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga ggt 768 Asp HisCys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg Gly 245 250 255 tcaact tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa atg 816 Ser ThrTyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met 260 265 270 acttta act gta tta gat cta att gta ctt ttc cca ttt tat gat att 864 Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp Ile 275 280 285 cggtta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att ttt 912 Arg LeuTyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile Phe 290 295 300 acggat cca att ttt tca ctt aat act ctt cag gag tat gga cca act 960 Thr AspPro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro Thr 305 310 315 320ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat tat 1008 PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp Tyr 325 330 335tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt ggg 1056 LeuGln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe Gly 340 345 350aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga cct 1104 LysAsp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro 355 360 365agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat aaa 1152 SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys 370 375 380tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt tat 1200 SerThr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr 385 390 395400 cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag gta 1248Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys Val 405 410415 tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa aaa 1296Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln Lys 420 425430 aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc cat 1344Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly His 435 440445 gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca gat 1392Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp 450 455460 gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa tgt 1440Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys 465 470475 480 ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg aca1488 Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr 485490 495 cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att act1536 His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr 500505 510 caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc att1584 Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile 515520 525 att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa gaa1632 Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu 530535 540 tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca gcc1680 Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala Ala 545550 555 560 ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc actaac 1728 Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn565 570 575 tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atctac 1776 Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr580 585 590 att aat aaa act atg aat aaa gat gat gat tta aca tat caa acattt 1824 Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr Phe595 600 605 gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aagaat 1872 Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys Asn610 615 620 gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atctat 1920 Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr625 630 635 640 ata gat aag ata gaa ttt atc cca gta caa ttg taa 1956 IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 52 <211>LENGTH: 651 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <400>SEQUENCE: 52 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val ThrPro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe LeuArg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr ValLys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr GlnSer Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala PheMet Ala Gln 100 105 110 Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu TyrAla Lys Ser Lys 115 120 125 Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn AsnPhe Glu Asp Tyr Val 130 135 140 Asn Ala Leu Asn Ser Trp Lys Lys Thr ProLeu Ser Leu Arg Ser Lys 145 150 155 160 Arg Ser Gln Asp Arg Ile Arg GluLeu Phe Ser Gln Ala Glu Ser His 165 170 175 Phe Arg Asn Ser Met Pro SerPhe Ala Val Ser Lys Phe Glu Val Leu 180 185 190 Phe Leu Pro Thr Tyr AlaGln Ala Ala Asn Thr His Leu Leu Leu Leu 195 200 205 Lys Asp Ala Gln ValPhe Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp 210 215 220 Val Ala Glu PheTyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr 225 230 235 240 Asp HisCys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg Gly 245 250 255 SerThr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met 260 265 270Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp Ile 275 280285 Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile Phe 290295 300 Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro Thr305 310 315 320 Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu PheAsp Tyr 325 330 335 Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro GlyTyr Phe Gly 340 345 350 Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr ValGlu Thr Arg Pro 355 360 365 Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp Lys 370 375 380 Ser Thr Glu Pro Val Gln Lys Leu Ser PheAsp Gly Gln Lys Val Tyr 385 390 395 400 Arg Thr Ile Ala Asn Thr Asp ValAla Ala Trp Pro Asn Gly Lys Val 405 410 415 Tyr Leu Gly Val Thr Lys ValAsp Phe Ser Gln Tyr Asp Asp Gln Lys 420 425 430 Asn Glu Thr Ser Thr GlnThr Tyr Asp Ser Lys Arg Asn Asn Gly His 435 440 445 Val Ser Ala Gln AspSer Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp 450 455 460 Glu Pro Leu GluLys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys 465 470 475 480 Phe LeuMet Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr 485 490 495 HisArg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr 500 505 510Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile 515 520525 Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu 530535 540 Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala Ala545 550 555 560 Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser ThrThr Asn 565 570 575 Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe LeuVal Ile Tyr 580 585 590 Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu ThrTyr Gln Thr Phe 595 600 605 Asp Leu Ala Thr Thr Asn Ser Asn Met Gly PheSer Gly Asp Lys Asn 610 615 620 Glu Leu Ile Ile Gly Ala Glu Ser Phe ValSer Asn Glu Lys Ile Tyr 625 630 635 640 Ile Asp Lys Ile Glu Phe Ile ProVal Gln Leu 645 650 <210> SEQ ID NO 53 <211> LENGTH: 1959 <212> TYPE:DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 53 atg aat cca aac aatcga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caaact aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Gln ThrAsn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaagaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu GluLeu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaagtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gttgta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val ValGly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctcact tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu ThrSer Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gat gct gaccca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp ProTrp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gat aag aaaata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cag ggt cttcaa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu GlnAsn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaaaca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys ThrPro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa gat cga ata agggaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg GluLeu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tcattt gca gtt tcc gga ttc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser PheAla Val Ser Gly Phe Glu Val 180 185 190 ctg ttt cta cca aca tat gca caagct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gtt ttt ggagaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly GluGlu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tat cat agacaa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg GlnLeu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gtt aat tggtat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gat gca tgggtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp ValLys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gta tta gatcta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp LeuIle Val Leu Phe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggggtt aaa aca gaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gat cca att ttt tcactt aat act ctt cag gag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser LeuAsn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaaaac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu AsnSer Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag ggg att gaattt cat acg cgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aattat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn TyrTrp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agt ata gga tct agtaag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser LysThr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gtacaa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val GlnLys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga act ata gctaat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gttacg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val Tyr Leu Gly Val ThrLys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agtaca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser ThrGln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agt gca caggat tct att gac caa tta ccg cca gaa aca aca 1392 His Val Ser Ala Gln AspSer Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaaaaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu LysAla Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atgcag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cat aga agtgta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His Arg Ser ValAsp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caa ctt ccagta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro ValVal Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggtcca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aattca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn SerIle Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg ttacaa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala Leu Leu GlnArg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cgactt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tac att aataaa act atg aat aaa gat gat gat tta aca tat caa aca 1824 Tyr Ile Asn LysThr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctcgca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu AlaThr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa cttata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tat atagat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 54 <211> LENGTH: 652<212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 54 MetAsn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 7580 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 9095 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100105 110 \ 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn AsnPhe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr ProLeu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg GluLeu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro SerPhe Ala Val Ser Gly Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr AlaGln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln ValPhe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu PheTyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp HisCys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly SerThr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 MetThr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295300 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu PheAsp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro GlyTyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr ValGlu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser PheAsp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp ValAla Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys ValAsp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr GlnThr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala Gln AspSer Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu GluLys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe LeuMet Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr HisArg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 ThrGln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser ThrThr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe LeuVal Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu ThrTyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly PheSer Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe ValSer Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile ProVal Gln Leu 645 650 <210> SEQ ID NO 55 <211> LENGTH: 1956 <212> TYPE:DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(1953) <400> SEQUENCE: 55 atg aat cca aac aatcga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caaact aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Gln ThrAsn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaagaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu GluLeu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaagtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gttgta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val ValGly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctcact tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu ThrSer Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gaa gac ccatgg aag gct ttt atg gca caa 336 Asn Thr Ile Trp Pro Ser Glu Asp Pro TrpLys Ala Phe Met Ala Gln 100 105 110 gtt gaa gta ctg ata gat aag aaa atagag gag tat gct aaa agt aaa 384 Val Glu Val Leu Ile Asp Lys Lys Ile GluGlu Tyr Ala Lys Ser Lys 115 120 125 gct ctt gca gag tta cag ggt ctt caaaat aat ttc gaa gat tat gtt 432 Ala Leu Ala Glu Leu Gln Gly Leu Gln AsnAsn Phe Glu Asp Tyr Val 130 135 140 aat gcg tta aat tcc tgg aag aaa acacct tta agt ttg cga aat cca 480 Asn Ala Leu Asn Ser Trp Lys Lys Thr ProLeu Ser Leu Arg Asn Pro 145 150 155 160 cac agc caa ggt cga ata agg gaactt ttt tct caa gca gaa agt cat 528 His Ser Gln Gly Arg Ile Arg Glu LeuPhe Ser Gln Ala Glu Ser His 165 170 175 ttt cgt aat tcc atg ccg tca tttgca gtt tcc aaa ttc gaa gtg ctg 576 Phe Arg Asn Ser Met Pro Ser Phe AlaVal Ser Lys Phe Glu Val Leu 180 185 190 ttt cta cca aca tat gca caa gctgca aat aca cat tta ttg cta tta 624 Phe Leu Pro Thr Tyr Ala Gln Ala AlaAsn Thr His Leu Leu Leu Leu 195 200 205 aaa gat gct caa gtt ttt gga gaagaa tgg gga tat tct tca gaa gat 672 Lys Asp Ala Gln Val Phe Gly Glu GluTrp Gly Tyr Ser Ser Glu Asp 210 215 220 gtt gct gaa ttt tat cat aga caatta aaa ctt aca caa caa tac act 720 Val Ala Glu Phe Tyr His Arg Gln LeuLys Leu Thr Gln Gln Tyr Thr 225 230 235 240 gac cat tgt gtt aat tgg tataat gtt gga tta aat ggt tta aga ggt 768 Asp His Cys Val Asn Trp Tyr AsnVal Gly Leu Asn Gly Leu Arg Gly 245 250 255 tca act tat gat gca tgg gtcaaa ttt aac cgt ttt cgc aga gaa atg 816 Ser Thr Tyr Asp Ala Trp Val LysPhe Asn Arg Phe Arg Arg Glu Met 260 265 270 act tta act gta tta gat ctaatt gta ctt ttc cca ttt tat gat att 864 Thr Leu Thr Val Leu Asp Leu IleVal Leu Phe Pro Phe Tyr Asp Ile 275 280 285 cgg tta tac tca aaa ggg gttaaa aca gaa cta aca aga gac att ttt 912 Arg Leu Tyr Ser Lys Gly Val LysThr Glu Leu Thr Arg Asp Ile Phe 290 295 300 acg gat cca att ttt tca cttaat act ctt cag gag tat gga cca act 960 Thr Asp Pro Ile Phe Ser Leu AsnThr Leu Gln Glu Tyr Gly Pro Thr 305 310 315 320 ttt ttg agt ata gaa aactct att cga aaa cct cat tta ttt gat tat 1008 Phe Leu Ser Ile Glu Asn SerIle Arg Lys Pro His Leu Phe Asp Tyr 325 330 335 tta cag ggg att gaa tttcat acg cgt ctt caa cct ggt tac ttt ggg 1056 Leu Gln Gly Ile Glu Phe HisThr Arg Leu Gln Pro Gly Tyr Phe Gly 340 345 350 aaa gat tct ttc aat tattgg tct ggt aat tat gta gaa act aga cct 1104 Lys Asp Ser Phe Asn Tyr TrpSer Gly Asn Tyr Val Glu Thr Arg Pro 355 360 365 agt ata gga tct agt aagaca att act tcc cca ttt tat gga gat aaa 1152 Ser Ile Gly Ser Ser Lys ThrIle Thr Ser Pro Phe Tyr Gly Asp Lys 370 375 380 tct act gaa cct gta caaaag cta agc ttt gat gga caa aaa gtt tat 1200 Ser Thr Glu Pro Val Gln LysLeu Ser Phe Asp Gly Gln Lys Val Tyr 385 390 395 400 cga act ata gct aataca gac gta gcg gct tgg ccg aat ggt aag gta 1248 Arg Thr Ile Ala Asn ThrAsp Val Ala Ala Trp Pro Asn Gly Lys Val 405 410 415 tat tta ggt gtt acgaaa gtt gat ttt agt caa tat gat gat caa aaa 1296 Tyr Leu Gly Val Thr LysVal Asp Phe Ser Gln Tyr Asp Asp Gln Lys 420 425 430 aat gaa act agt acacaa aca tat gat tca aaa aga aac aat ggc cat 1344 Asn Glu Thr Ser Thr GlnThr Tyr Asp Ser Lys Arg Asn Asn Gly His 435 440 445 gta agt gca cag gattct att gac caa tta ccg cca gaa aca aca gat 1392 Val Ser Ala Gln Asp SerIle Asp Gln Leu Pro Pro Glu Thr Thr Asp 450 455 460 gaa cca ctt gaa aaagca tat agt cat cag ctt aat tac gcg gaa tgt 1440 Glu Pro Leu Glu Lys AlaTyr Ser His Gln Leu Asn Tyr Ala Glu Cys 465 470 475 480 ttc tta atg caggac cgt cgt gga aca att cca ttt ttt act tgg aca 1488 Phe Leu Met Gln AspArg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr 485 490 495 cat aga agt gtagac ttt ttt aat aca att gat gct gaa aag att act 1536 His Arg Ser Val AspPhe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr 500 505 510 caa ctt cca gtagtg aaa gca tat gcc ttg tct tca ggt gct tcc att 1584 Gln Leu Pro Val ValLys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile 515 520 525 att gaa ggt ccagga ttc aca gga gga aat tta cta ttc cta aaa gaa 1632 Ile Glu Gly Pro GlyPhe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu 530 535 540 tct agt aat tcaatt gct aaa ttt aaa gtt aca tta aat tca gca gcc 1680 Ser Ser Asn Ser IleAla Lys Phe Lys Val Thr Leu Asn Ser Ala Ala 545 550 555 560 ttg tta caacga tat cgt gta aga ata cgc tat gct tct acc act aac 1728 Leu Leu Gln ArgTyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn 565 570 575 tta cga cttttt gtg caa aat tca aac aat gat ttt ctt gtc atc tac 1776 Leu Arg Leu PheVal Gln Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr 580 585 590 att aat aaaact atg aat aaa gat gat gat tta aca tat caa aca ttt 1824 Ile Asn Lys ThrMet Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr Phe 595 600 605 gat ctc gcaact act aat tct aat atg ggg ttc tcg ggt gat aag aat 1872 Asp Leu Ala ThrThr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys Asn 610 615 620 gaa ctt ataata gga gca gaa tct ttc gtt tct aat gaa aaa atc tat 1920 Glu Leu Ile IleGly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr 625 630 635 640 ata gataag ata gaa ttt atc cca gta caa ttg taa 1956 Ile Asp Lys Ile Glu Phe IlePro Val Gln Leu 645 650 <210> SEQ ID NO 56 <211> LENGTH: 651 <212> TYPE:PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 56 Met Asn ProAsn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 GlyVal Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 AsnThr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Ala Gln 100 105 110Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys 115 120125 Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val 130135 140 Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Asn Pro145 150 155 160 His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala GluSer His 165 170 175 Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val Leu 180 185 190 Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr HisLeu Leu Leu Leu 195 200 205 Lys Asp Ala Gln Val Phe Gly Glu Glu Trp GlyTyr Ser Ser Glu Asp 210 215 220 Val Ala Glu Phe Tyr His Arg Gln Leu LysLeu Thr Gln Gln Tyr Thr 225 230 235 240 Asp His Cys Val Asn Trp Tyr AsnVal Gly Leu Asn Gly Leu Arg Gly 245 250 255 Ser Thr Tyr Asp Ala Trp ValLys Phe Asn Arg Phe Arg Arg Glu Met 260 265 270 Thr Leu Thr Val Leu AspLeu Ile Val Leu Phe Pro Phe Tyr Asp Ile 275 280 285 Arg Leu Tyr Ser LysGly Val Lys Thr Glu Leu Thr Arg Asp Ile Phe 290 295 300 Thr Asp Pro IlePhe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro Thr 305 310 315 320 Phe LeuSer Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp Tyr 325 330 335 LeuGln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe Gly 340 345 350Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro 355 360365 Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys 370375 380 Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr385 390 395 400 Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn GlyLys Val 405 410 415 Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr AspAsp Gln Lys 420 425 430 Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys ArgAsn Asn Gly His 435 440 445 Val Ser Ala Gln Asp Ser Ile Asp Gln Leu ProPro Glu Thr Thr Asp 450 455 460 Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu Cys 465 470 475 480 Phe Leu Met Gln Asp Arg Arg GlyThr Ile Pro Phe Phe Thr Trp Thr 485 490 495 His Arg Ser Val Asp Phe PheAsn Thr Ile Asp Ala Glu Lys Ile Thr 500 505 510 Gln Leu Pro Val Val LysAla Tyr Ala Leu Ser Ser Gly Ala Ser Ile 515 520 525 Ile Glu Gly Pro GlyPhe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu 530 535 540 Ser Ser Asn SerIle Ala Lys Phe Lys Val Thr Leu Asn Ser Ala Ala 545 550 555 560 Leu LeuGln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn 565 570 575 LeuArg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr 580 585 590Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr Phe 595 600605 Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys Asn 610615 620 Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr625 630 635 640 Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650<210> SEQ ID NO 57 <211> LENGTH: 1956 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1953) <400> SEQUENCE: 57 atg aat cca aac aat cga agt gaa cat gatacg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caatat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaagaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tctaca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att ttaggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caatca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln SerPhe Leu 85 90 95 aac act ata tgg cca agt gaa gac cca tgg aag gct ttt atggca caa 336 Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met AlaGln 100 105 110 gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaaagt aaa 384 Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys SerLys 115 120 125 gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gattat gtt 432 Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp TyrVal 130 135 140 aat gcg tta aat tcc tgg aag aaa ttt cac cat tct cgt cgttct aaa 480 Asn Ala Leu Asn Ser Trp Lys Lys Phe His His Ser Arg Arg SerLys 145 150 155 160 aga agc caa gat cga ata agg gaa ctt ttt tct caa gcagaa agt cat 528 Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala GluSer His 165 170 175 ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttcgaa gtg ctg 576 Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe GluVal Leu 180 185 190 ttt cta cca aca tat gca caa gct gca aat aca cat ttattg cta tta 624 Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu LeuLeu Leu 195 200 205 aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tcttca gaa gat 672 Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser SerGlu Asp 210 215 220 gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caacaa tac act 720 Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln GlnTyr Thr 225 230 235 240 gac cat tgt gtt aat tgg tat aat gtt gga tta aatggt tta aga ggt 768 Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn GlyLeu Arg Gly 245 250 255 tca act tat gat gca tgg gtc aaa ttt aac cgt tttcgc aga gaa atg 816 Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe ArgArg Glu Met 260 265 270 act tta act gta tta gat cta att gta ctt ttc ccattt tat gat att 864 Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro PheTyr Asp Ile 275 280 285 cgg tta tac tca aaa ggg gtt aaa aca gaa cta acaaga gac att ttt 912 Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr ArgAsp Ile Phe 290 295 300 acg gat cca att ttt tca ctt aat act ctt cag gagtat gga cca act 960 Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu TyrGly Pro Thr 305 310 315 320 ttt ttg agt ata gaa aac tct att cga aaa cctcat tta ttt gat tat 1008 Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro HisLeu Phe Asp Tyr 325 330 335 tta cag ggg att gaa ttt cat acg cgt ctt caacct ggt tac ttt ggg 1056 Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln ProGly Tyr Phe Gly 340 345 350 aaa gat tct ttc aat tat tgg tct ggt aat tatgta gaa act aga cct 1104 Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr ValGlu Thr Arg Pro 355 360 365 agt ata gga tct agt aag aca att act tcc ccattt tat gga gat aaa 1152 Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro PheTyr Gly Asp Lys 370 375 380 tct act gaa cct gta caa aag cta agc ttt gatgga caa aaa gtt tat 1200 Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp GlyGln Lys Val Tyr 385 390 395 400 cga act ata gct aat aca gac gta gcg gcttgg ccg aat ggt aag gta 1248 Arg Thr Ile Ala Asn Thr Asp Val Ala Ala TrpPro Asn Gly Lys Val 405 410 415 tat tta ggt gtt acg aaa gtt gat ttt agtcaa tat gat gat caa aaa 1296 Tyr Leu Gly Val Thr Lys Val Asp Phe Ser GlnTyr Asp Asp Gln Lys 420 425 430 aat gaa act agt aca caa aca tat gat tcaaaa aga aac aat ggc cat 1344 Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser LysArg Asn Asn Gly His 435 440 445 gta agt gca cag gat tct att gac caa ttaccg cca gaa aca aca gat 1392 Val Ser Ala Gln Asp Ser Ile Asp Gln Leu ProPro Glu Thr Thr Asp 450 455 460 gaa cca ctt gaa aaa gca tat agt cat cagctt aat tac gcg gaa tgt 1440 Glu Pro Leu Glu Lys Ala Tyr Ser His Gln LeuAsn Tyr Ala Glu Cys 465 470 475 480 ttc tta atg cag gac cgt cgt gga acaatt cca ttt ttt act tgg aca 1488 Phe Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp Thr 485 490 495 cat aga agt gta gac ttt ttt aat acaatt gat gct gaa aag att act 1536 His Arg Ser Val Asp Phe Phe Asn Thr IleAsp Ala Glu Lys Ile Thr 500 505 510 caa ctt cca gta gtg aaa gca tat gccttg tct tca ggt gct tcc att 1584 Gln Leu Pro Val Val Lys Ala Tyr Ala LeuSer Ser Gly Ala Ser Ile 515 520 525 att gaa ggt cca gga ttc aca gga ggaaat tta cta ttc cta aaa gaa 1632 Ile Glu Gly Pro Gly Phe Thr Gly Gly AsnLeu Leu Phe Leu Lys Glu 530 535 540 tct agt aat tca att gct aaa ttt aaagtt aca tta aat tca gca gcc 1680 Ser Ser Asn Ser Ile Ala Lys Phe Lys ValThr Leu Asn Ser Ala Ala 545 550 555 560 ttg tta caa cga tat cgt gta agaata cgc tat gct tct acc act aac 1728 Leu Leu Gln Arg Tyr Arg Val Arg IleArg Tyr Ala Ser Thr Thr Asn 565 570 575 tta cga ctt ttt gtg caa aat tcaaac aat gat ttt ctt gtc atc tac 1776 Leu Arg Leu Phe Val Gln Asn Ser AsnAsn Asp Phe Leu Val Ile Tyr 580 585 590 att aat aaa act atg aat aaa gatgat gat tta aca tat caa aca ttt 1824 Ile Asn Lys Thr Met Asn Lys Asp AspAsp Leu Thr Tyr Gln Thr Phe 595 600 605 gat ctc gca act act aat tct aatatg ggg ttc tcg ggt gat aag aat 1872 Asp Leu Ala Thr Thr Asn Ser Asn MetGly Phe Ser Gly Asp Lys Asn 610 615 620 gaa ctt ata ata gga gca gaa tctttc gtt tct aat gaa aaa atc tat 1920 Glu Leu Ile Ile Gly Ala Glu Ser PheVal Ser Asn Glu Lys Ile Tyr 625 630 635 640 ata gat aag ata gaa ttt atccca gta caa ttg taa 1956 Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 58 <211> LENGTH: 651 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <400> SEQUENCE: 58 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn HisAsn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu LeuAsn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser ValVal Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser GluAsp Pro Trp Lys Ala Phe Met Ala Gln 100 105 110 Val Glu Val Leu Ile AspLys Lys Ile Glu Glu Tyr Ala Lys Ser Lys 115 120 125 Ala Leu Ala Glu LeuGln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val 130 135 140 Asn Ala Leu AsnSer Trp Lys Lys Phe His His Ser Arg Arg Ser Lys 145 150 155 160 Arg SerGln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His 165 170 175 PheArg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val Leu 180 185 190Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu Leu 195 200205 Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp 210215 220 Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr225 230 235 240 Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly LeuArg Gly 245 250 255 Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe ArgArg Glu Met 260 265 270 Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp Ile 275 280 285 Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu LeuThr Arg Asp Ile Phe 290 295 300 Thr Asp Pro Ile Phe Ser Leu Asn Thr LeuGln Glu Tyr Gly Pro Thr 305 310 315 320 Phe Leu Ser Ile Glu Asn Ser IleArg Lys Pro His Leu Phe Asp Tyr 325 330 335 Leu Gln Gly Ile Glu Phe HisThr Arg Leu Gln Pro Gly Tyr Phe Gly 340 345 350 Lys Asp Ser Phe Asn TyrTrp Ser Gly Asn Tyr Val Glu Thr Arg Pro 355 360 365 Ser Ile Gly Ser SerLys Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys 370 375 380 Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr 385 390 395 400 Arg ThrIle Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys Val 405 410 415 TyrLeu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln Lys 420 425 430Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly His 435 440445 Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp 450455 460 Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys465 470 475 480 Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe ThrTrp Thr 485 490 495 His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala GluLys Ile Thr 500 505 510 Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser SerGly Ala Ser Ile 515 520 525 Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu Lys Glu 530 535 540 Ser Ser Asn Ser Ile Ala Lys Phe Lys ValThr Leu Asn Ser Ala Ala 545 550 555 560 Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr Asn 565 570 575 Leu Arg Leu Phe Val Gln AsnSer Asn Asn Asp Phe Leu Val Ile Tyr 580 585 590 Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Gln Thr Phe 595 600 605 Asp Leu Ala Thr ThrAsn Ser Asn Met Gly Phe Ser Gly Asp Lys Asn 610 615 620 Glu Leu Ile IleGly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr 625 630 635 640 Ile AspLys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 59 <211>LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE:59 atg aat cca aac aat cga agt gaa cat gat acg ata aag gtt aca cct 48Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 aac agt gaa ttg caa act aac cat aat caa tat cct tta gct gac aat 96Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 actgaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gttggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val GlyThr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gttcca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val ProPhe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act atatgg cca agt gat gct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaagta ctg ata gat aag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu ValLeu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct cttgca gag tta cag ggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu AlaGlu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcgtta aat tcc tgg aag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa agaagc caa ggt cga ata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg SerGln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat tttcgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe ArgAsn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg tttcta cca aca tat gca caa gct gca aat aca cat tta ttg cta 624 Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaagat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gttgct gaa ttt tat cat aga caa tta aaa ctt aca caa caa tac 720 Asp Val AlaGlu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 actgac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr AspHis Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggttca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly SerThr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atgact tta act gta tta gat cta att gta ctt ttc cca ttt tat gat 864 Met ThrLeu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 attcgg tta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 tttacg gat cca att ttt acc ctt aat aca cta cag aag tac gga cca 960 Phe ThrAsp Pro Ile Phe Thr Leu Asn Thr Leu Gln Lys Tyr Gly Pro 305 310 315 320act ttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt gat 1008 ThrPhe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335tat tta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 GlyLys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365cct agt ata gga tct agt aag aca att act tcc cca ttt tat gga gat 1152 ProSer Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380aaa tct act gaa cct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 tat cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425430 aaa aat gaa act agt aca caa aca tat gat tca aaa aga aac aat ggc 1344Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 cat gta agt gca cag gat tct att gac caa tta ccg cca gaa aca aca 1392His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455460 gat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470475 480 tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485490 495 aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag att1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515520 525 att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530535 540 gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545550 555 560 gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct accact 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr565 570 575 aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtcatc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 tac att aat aaa act atg aat aaa gat gat gat tta aca tat caaaca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr595 600 605 ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gataag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys610 615 620 aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaaatc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO60 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin<400> SEQUENCE: 60 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile LysVal Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn SerThr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser PheTyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro TrpLys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly LeuGln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Gly ArgIle Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp ValAla Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 Phe Thr Asp Pro Ile Phe Thr Leu Asn Thr Leu Gln Lys TyrGly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg LeuGln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr IleThr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln LysLeu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly ValThr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val SerAla Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe LeuLys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu AsnSer Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg TyrAla Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp AspAsp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala GluSer Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 61 <211> LENGTH: 1959<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Recombinant delta endotoxin <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 61 atg aat ccaaac aat cga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro AsnAsn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaattg caa act aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu LeuGln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca acacta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tctacg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser ThrGlu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga atttct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile SerVal Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggggca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gatgct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp AlaAsp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gataag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp LysLys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cagggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tggaag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa ggt cgaata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Gly Arg IleArg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atgccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met ProSer Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta cca aca tatgca caa gct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr AlaGln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gttttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val PheGly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tatcat aga caa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gttaat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val AsnTrp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gatgca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp AlaTrp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gtatta gat cta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 gtt cgg tta tac ccaaaa ggg gtt aaa aca gaa cta aca aga gac att 912 Val Arg Leu Tyr Pro LysGly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 tct acg gat cca attttt gcc gtt aat act ctg tgg gaa tac gga cca 960 Ser Thr Asp Pro Ile PheAla Val Asn Thr Leu Trp Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agtata gaa aac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag gggatt gaa ttt cat acg cgt ctt cga cct ggt tac ttt 1056 Tyr Leu Gln Gly IleGlu Phe His Thr Arg Leu Arg Pro Gly Tyr Phe 340 345 350 ggg aaa gat tctttc aat tat tgg tct ggt aat tat gca gaa act aga 1104 Gly Lys Asp Ser PheAsn Tyr Trp Ser Gly Asn Tyr Ala Glu Thr Arg 355 360 365 cct agt ata ggatct agt aag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaacct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga actata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr IleAla Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gta tat ttaggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaaact agt aca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agtgca cag gat tct att gac caa tta ccg cca gaa aca aca 1392 His Val Ser AlaGln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa ccactt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttctta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe LeuMet Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cataga agt gta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His ArgSer Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caactt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att attgaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile GluGly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tctagt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser SerAsn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gccttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aactta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776 Asn LeuArg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tacatt aat aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824 Tyr IleAsn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 tttgat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aatgaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn GluLeu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp LysIle Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 62 <211> LENGTH:652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 62Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 2530 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 4045 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 5560 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 7075 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe GluAsp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 Lys Arg Ser Gln Gly Arg Ile Arg Glu Leu PheSer Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe AlaVal Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe GlyGlu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys ValAsn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr TyrAsp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Val ArgLeu Tyr Pro Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 SerThr Asp Pro Ile Phe Ala Val Asn Thr Leu Trp Glu Tyr Gly Pro 305 310 315320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Arg Pro Gly Tyr Phe340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Ala Glu ThrArg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp GlyGln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp PheSer Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser IleAsp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys AlaTyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg SerVal Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile IleGlu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 GluSer Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr GlnThr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser GlyAsp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser AsnGlu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 63 <211> LENGTH: 1959 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1956) <400> SEQUENCE: 63 atg aat cca aac aat cga agt gaacat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cataat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His AsnGln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aattat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn TyrLys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gacaac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cagatt tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttttat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe TyrGln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aaggct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys AlaPhe Met Ala 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gagtat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu TyrAla Lys Ser 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aatttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn PheGlu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct ttaagt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttttct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe SerGln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtttcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val SerLys Phe Glu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aataca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgggga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp GlyTyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaactt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys LeuThr Gln Gln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gttgga tta aat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa tttaac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe AsnArg Phe Arg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gtactt ttc cca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val LeuPhe Pro Phe Tyr Asp 275 280 285 gtt cgg tta tac cca aaa ggg gtt aaa acagaa cta aca aga gac att 912 Val Arg Leu Tyr Pro Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 ttt acg gat cca att ttt tca ctt aat actctt cag gag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr LeuGln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct attcga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile ArgLys Pro His Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acgcgt ctt cga cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Arg Pro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tctggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca attact tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile ThrSer Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag ctaagc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gacgta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp ValAla Ala Trp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gttgat ttt agt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val AspPhe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa acatat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct attgac caa tta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile AspGln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tatagt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr SerHis Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgtcgt gga aca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac tttttt aat aca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe PheAsn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaagca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys AlaTyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttcaca gga gga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gctaaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala LysPhe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tatcgt gta aga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr ArgVal Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtgcaa aat tca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atgaat aaa gat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act actaat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr AsnSer Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata ggagca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag atagaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile ProVal Gln Leu 645 650 <210> SEQ ID NO 64 <211> LENGTH: 652 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 64 Met Asn ProAsn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 GlyVal Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 AsnThr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser LysPhe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu TrpGly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln LeuLys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala TrpVal Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Val Arg Leu Tyr ProLys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp ProIle Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Arg Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro AsnGly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser LysArg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser HisGln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp PhePhe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val ValLys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser AsnSer Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 AsnLeu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650<210> SEQ ID NO 65 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1956) <400> SEQUENCE: 65 atg aat cca aac aat cga agt gaa cat gatacg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caatat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaagaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tctaca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att ttaggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caatca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln SerPhe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct tttatg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe MetAla 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gctaaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaagat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttgcga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer 145 150 155 160 aaa aga agc caa ggt cga ata agg gaa ctt ttt tct caagca gaa agt 528 Lys Arg Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaattc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cattta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tattct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt acacaa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr GlnGln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga ttaaat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu AsnGly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgtttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttccca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa ctaaca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile 290 295 300 ttt acg gat cca att ttt tta ctt aat act ctt caggag tat gga cca 960 Phe Thr Asp Pro Ile Phe Leu Leu Asn Thr Leu Gln GluTyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaacct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt cttcaa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu GlnPro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aattat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcccca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc tttgat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcggct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat tttagt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gattca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caatta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt catcag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt ggaaca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly ThrIle Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aataca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tatgcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca ggagga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa tttaaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gtaaga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aattca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaagat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tctaat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaatct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa tttatc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 66 <211> LENGTH: 652 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <400> SEQUENCE: 66 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu GlnThr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser SerThr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro PheAla Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val GluVal Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys AlaLeu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 ValAsn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155160 Lys Arg Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu LeuLeu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu ThrGln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys PheAsn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu IleVal Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe LeuLeu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln GlyIle Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn AsnGly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro GluThr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala TyrAla Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile AlaLys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln ArgTyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile AsnLys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 AsnGlu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ IDNO 67 <211> LENGTH: 1959 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Recombinant deltaendotoxin <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1956)<400> SEQUENCE: 67 atg aat cca aac aat cga agt gaa cat gat acg ata aaggtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys ValThr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caa tat cct ttagct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaa gaa ttt ttaaga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu ArgMet 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tct aca gta aaagat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att tta ggt gtt gta240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 6570 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caa tca ttt ctt288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct ttt atg gca336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gct aaa agt384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaa gat tat432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttg cga agt480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaaagt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaagtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta ttgcta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tcagaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caatac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga tta aat ggttta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly LeuArg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgt ttt cgcaga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg ArgGlu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttc cca ttttat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe TyrAsp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa cta aca agagac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg AspIle 290 295 300 ttt acg gat cca att ttt tca ctt aat act ctt cag gag tatgga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr GlyPro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaa cct cattta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt ctt cga cctggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Arg Pro GlyTyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aat tat gtagaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val GluThr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcc cca ttttat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc ttt gat ggacaa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly GlnLys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcg gct tggccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp ProAsn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat ttt agt caatat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gat tca aaaaga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys ArgAsn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caa tta ccgcca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro ProGlu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt cat cag cttaat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt gga aca attcca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile ProPhe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aat aca attgat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile AspAla Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tat gcc ttgtct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca gga gga aattta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa ttt aaa gttaca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val ThrLeu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gta aga atacgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aat tca aacaat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaa gat gatgat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp AspLeu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tct aat atgggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaa tct ttcgtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe ValSer Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa ttt atc ccagta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 68 <211> LENGTH: 652 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <400> SEQUENCE: 68 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn HisAsn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu LeuAsn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser ValVal Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser AspAla Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu IleAsp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala GluLeu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys ArgSer Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 HisPhe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn GlyLeu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu PhePro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn ThrLeu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn SerIle Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Arg Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe AsnTyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr GluPro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr ArgThr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 ValTyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe PheThr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp AlaGlu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu SerSer Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly AsnLeu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg ValArg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr MetAsn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala ThrThr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 69 <211>LENGTH: 1482 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1479) <400> SEQUENCE:69 agt aaa aga agc caa gat cga ata agg gaa ctt ttt tct caa gca gaa 48Ser Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 1 5 1015 agt cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaa ttc gaa 96Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu 20 25 30gtg ctg ttt cta cca aca tat gca caa gct gca aat aca cat tta ttg 144 ValLeu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 35 40 45 ctatta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tat tct tca 192 Leu LeuLys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 50 55 60 gaa gatgtt gct gaa ttt tat cat aga caa tta aaa ctt aca caa caa 240 Glu Asp ValAla Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln 65 70 75 80 tac actgac cat tgt gtt aat tgg tat aat gtt gga tta aat ggt tta 288 Tyr Thr AspHis Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 85 90 95 aga ggt tcaact tat gat gca tgg gtc aaa ttt aac cgt ttt cgc aga 336 Arg Gly Ser ThrTyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 100 105 110 gaa atg acttta act gta tta gat cta att gta ctt ttc cca ttt tat 384 Glu Met Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr 115 120 125 gat att cggtta tac tca aaa ggg gtt aaa aca gaa cta aca aga gac 432 Asp Ile Arg LeuTyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp 130 135 140 att ttt acggat cca att ttt tca ctt aat act ctt cag gag tat gga 480 Ile Phe Thr AspPro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly 145 150 155 160 cca actttt ttg agt ata gaa aac tct att cga aaa cct cat tta ttt 528 Pro Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe 165 170 175 gat tattta cag ggg att gaa ttt cat acg cgt ctt caa cct ggt tac 576 Asp Tyr LeuGln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr 180 185 190 ttt gggaaa gat tct ttc aat tat tgg tct ggt aat tat gta gaa act 624 Phe Gly LysAsp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr 195 200 205 aga cctagt ata gga tct agt aag aca att act tcc cca ttt tat gga 672 Arg Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly 210 215 220 gat aaatct act gaa cct gta caa aag cta agc ttt gat gga caa aaa 720 Asp Lys SerThr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys 225 230 235 240 gtttat cga act ata gct aat aca gac gta gcg gct tgg ccg aat ggt 768 Val TyrArg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly 245 250 255 aaggta tat tta ggt gtt acg aaa gtt gat ttt agt caa tat gat gat 816 Lys ValTyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 260 265 270 caaaaa aat gaa act agt aca caa aca tat gat tca aaa aga aac aat 864 Gln LysAsn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 275 280 285 ggccat gta agt gca cag gat tct att gac caa tta ccg cca gaa aca 912 Gly HisVal Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 290 295 300 acagat gaa cca ctt gaa aaa gca tat agt cat cag ctt aat tac gcg 960 Thr AspGlu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 305 310 315 320gaa tgt ttc tta atg cag gac cgt cgt gga aca att cca ttt ttt act 1008 GluCys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 325 330 335tgg aca cat aga agt gta gac ttt ttt aat aca att gat gct gaa aag 1056 TrpThr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 340 345 350att act caa ctt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct 1104 IleThr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala 355 360 365tcc att att gaa ggt cca gga ttc aca gga gga aat tta cta ttc cta 1152 SerIle Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu 370 375 380aaa gaa tct agt aat tca att gct aaa ttt aaa gtt aca tta aat tca 1200 LysGlu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser 385 390 395400 gca gcc ttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc 1248Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr 405 410415 act aac tta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc 1296Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val 420 425430 atc tac att aat aaa act atg aat aaa gat gat gat tta aca tat caa 1344Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln 435 440445 aca ttt gat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat 1392Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp 450 455460 aag aat gaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa 1440Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys 465 470475 480 atc tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1482 IleTyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 485 490 SEQ ID NO 70LENGTH: 493 TYPE: PRT ORGANISM: Artificial sequence FEATURE: OTHERINFORMATION: Recombinant delta endotoxin SEQUENCE: 70 Ser Lys Arg SerGln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 1 5 10 15 Ser His PheArg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu 20 25 30 Val Leu PheLeu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 35 40 45 Leu Leu LysAsp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 50 55 60 Gla Asp ValAla Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln 65 70 75 80 Tyr ThrAsp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 85 90 95 Arg GlySer Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 100 105 110 GluMet Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr 115 120 125Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp 130 135140 Ile Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly 145150 155 160 Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe 165 170 175 Asp Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln ProGly Tyr 180 185 190 Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr 195 200 205 Arg Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr SerPro Phe Tyr Gly 210 215 220 Asp Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys 225 230 235 240 Val Tyr Arg Thr Ile Ala Asn Thr AspVal Ala Ala Trp Pro Asn Gly 245 250 255 Lys Val Tyr Leu Gly Val Thr LysVal Asp Phe Ser Gln Tyr Asp Asp 260 265 270 Gln Lys Asn Glu Thr Ser ThrGln Thr Tyr Asp Ser Lys Arg Asn Asn 275 280 285 Gly His Val Ser Ala GlnAsp Ser Ile Asp Gln Leu Pro Pro Glu Thr 290 295 300 Thr Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 305 310 315 320 Glu Cys PheLeu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 325 330 335 Trp ThrHis Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 340 345 350 IleThr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala 355 360 365Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu 370 375380 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser 385390 395 400 Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala SerThr 405 410 415 Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp PheLeu Val 420 425 430 Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp LeuThr Tyr Gln 435 440 445 Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp 450 455 460 Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser PheVal Ser Asn Glu Lys 465 470 475 480 Ile Tyr Ile Asp Lys Ile Glu Phe IlePro Val Gln Leu 485 490 SEQ ID NO 71 LENGTH: 23 TYPE: DNA ORGANISM:Artificial sequence FEATURE: OTHER INFORMATION: SyntheticOligonucleotide SEQUENCE: 71 agacaactct acagtaaaag atg 23 SEQ ID NO 72LENGTH: 20 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHERINFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 72 ggtaattggtcaatagaatc 20 <210> SEQ ID NO 73 <211> LENGTH: 39 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Oligonucleotide <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (21)..(23) <223> OTHER INFORMATION: N = A, T, G, C (25%each) <400> SEQUENCE: 73 cagaagatgt tgctgaattc nnncatagac aattaaaac 39<210> SEQ ID NO 74 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (19)..(21) <223> OTHER INFORMATION: N = A, T, G, C (25% each)<400> SEQUENCE: 74 gatgttgctg aattctatnn nagacaatta aaac 34 <210> SEQ IDNO 75 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (17)..(17) <223> OTHER INFORMATION: N = A, T, C (16% each); G(52%) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(18)..(18) <223> OTHER INFORMATION: N = T, G, C (10% each); A (70%)<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19)<223> OTHER INFORMATION: N = A, T, G, C (25% each) <400> SEQUENCE: 75cccattttat gatattnnnt tatactcaaa agg 33 <210> SEQ ID NO 76 <211> LENGTH:64 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (24)..(24) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (25)..(25) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (26)..(26) <223> OTHERINFORMATION: N = A, T, G, C (25% each) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (27)..(28) <223> OTHER INFORMATION: N = A,T, G (6%) each); C (82%) <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (29)..(29) <223> OTHER INFORMATION: N = A, T, G, C (25%each) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(30)..(30) <223> OTHER INFORMATION: N = A, T, G (6% each); C (82%) <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (31)..(31) <223>OTHER INFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (32)..(32) <223> OTHERINFORMATION: N = A, T, G, C (25%) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (33)..(33) <223> OTHER INFORMATION: N = A,G, C (6% each); T (82%) <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (34)..(34) <223> OTHER INFORMATION: N = A, T, G (6%each) ; C (82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (35)..(35) <223> OTHER INFORMATION: N = A, G, C (6% each); T(82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(36)..(36) <223> OTHER INFORMATION: N = A, T, G (6% each) ; C (82%)<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (37)..(37)<223> OTHER INFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (38)..(38) <223> OTHERINFORMATION: N = A, T, G, C (25% each) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (39)..(39) <223> OTHER INFORMATION: N = A,T, G (6% each); C (82%) <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (40)..(40) <223> OTHER INFORMATION: N = A, T, C (6%each); G (82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (41)..(41) <223> OTHER INFORMATION: N = A, T, G, C (25% each)<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (42)..(42)<223> OTHER INFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (43)..(43) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (44)..(44) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <400> SEQUENCE: 76agctatgctg gtctcggaag aaannnnnnn nnnnnnnnnn nnnnaaaaga agccaagatc 60gaat 64 <210> SEQ ID NO 77 <211> LENGTH: 40 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Oligonucleotide <400> SEQUENCE: 77 ggtcacctag gtctctcttccaggaattta acgcattaac 40 <210> SEQ ID NO 78 <211> LENGTH: 65 <212> TYPE:DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (22)..(22) <223> OTHER INFORMATION: N = A,G, C (6% each); T (82%) <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (23)..(23) <223> OTHER INFORMATION: N = T, G, C (6%each); A (82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (24)..(24) <223> OTHER INFORMATION: N = A, T, G (1% each); C(97%) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(25)..(25) <223> OTHER INFORMATION: N = A, T, C (6% each); G (82%) <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (26)..(26) <223>OTHER INFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (27)..(27) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (28)..(28) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (29)..(29) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (30)..(30) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (31)..(31) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (32)..(33) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (34)..(34) <223> OTHERINFORMATION: N = A, T, G (15% each); C (55%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (35)..(36) <223> OTHERINFORMATION: N = A, G, C (15% each); T (55%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (37)..(37) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (38)..(38) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (39)..(39) <223> OTHERINFORMATION: N = A, T, G (1% each); C (97%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (40)..(40) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (41)..(41) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (42)..(42) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (43)..(44) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (45)..(45) <223> OTHERINFORMATION: N = A, T, G, C (25% each) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (46)..(48) <223> OTHER INFORMATION: N = A,T, C (6% each); G (82%) <400> SEQUENCE: 78 agctatgctg gtctcccatttnnnnnnnnn nnnnnnnnnn nnnnnnnngt taaaacagaa 60 ctaac 65 <210> SEQ ID NO79 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide <400>SEQUENCE: 79 atccagtggg gtctcaaatg ggaaaagtac aattag 36 <210> SEQ ID NO80 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (23)..(23) <223>OTHER INFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (24)..(26) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (27)..(27) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (28)..(28) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (29)..(30) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (31)..(31) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (32)..(32) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (33)..(33) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (34)..(34) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (35)..(35) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (36)..(36) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (37)..(37) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (38)..(38) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (39)..(39) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (40)..(40) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (41)..(41) <223> OTHERINFORMATION: N = A, C (8% each); T (1%); G (83%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (42)..(42) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (43)..(43) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (44)..(44) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (45)..(45) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (46)..(46) <223> OTHERINFORMATION: N = A, T,G (1% each); C (97%) <400> SEQUENCE: 80 catttttacggatccaattt ttnnnnnnnn nnnnnnnnnn nnnnnnggac caactttttt 60 gag 63 <210>SEQ ID NO 81 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (28)..(28) <223> OTHER INFORMATION: N = A, G, C (6% each); T(82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(29)..(29) <223> OTHER INFORMATION: N = T, G, C (6% each); A (82%) <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (30)..(30) <223>OTHER INFORMATION: N = A, T, G (1% each); C (97%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (31)..(33) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (34)..(35) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (36)..(36) <223> OTHERINFORMATION: N = A, T, G, C (25% each) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (37)..(37) <223> OTHER INFORMATION: N = A(82%); T (2%); G, C (8% each) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (38)..(39) <223> OTHER INFORMATION: N = T,G, C (6% each); A (82%) <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (40)..(40) <223> OTHER INFORMATION: N = A, T, C (6%each); G (82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (41)..(41) <223> OTHER INFORMATION: N = T, G, C (6% each); A(82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(42)..(42) <223> OTHER INFORMATION: N = A, G, C (6% each); T (82%) <400>SEQUENCE: 81 gaatttcata cgcgtcttca acctggtnnn nnnnnnnnnn nntctttcaattattggtct 60 gg 62 <210> SEQ ID NO 82 <211> LENGTH: 73 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (41)..(41) <223> OTHER INFORMATION: N = A,G, C (6% each); T (82%) <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (42)..(43) <223> OTHER INFORMATION: N = A (0%); T,C (9%each); G (82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (44)..(45) <223> OTHER INFORMATION: N = A, T, G (6% each); C(82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(46)..(46) <223> OTHER INFORMATION: N = A, T, G, C (25% each) <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (47)..(48) <223>OTHER INFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (49)..(49) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (50)..(51) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (52)..(52) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (53)..(54) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (55)..(55) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <400> SEQUENCE: 82aaaagtttat cgaactatag ctaatacaga cgtagcggct nnnnnnnnnn nnnnngtata 60tttaggtgtt acg 73 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 83 ggagttccatttgctggggc 20 <210> SEQ ID NO 84 <211> LENGTH: 17 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Oligonucleotide <400> SEQUENCE: 84 atctccataa aatgggg 17 <210>SEQ ID NO 85 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <400> SEQUENCE: 85 gcgaagtaaa agaagccaag gtcgaataag gg32 <210> SEQ ID NO 86 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <400> SEQUENCE: 86 cctttaagtt tgcgaaatcc acacagccaaggtcgaataa ggg 43 <210> SEQ ID NO 87 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 87 cccattttatgatgttcggt tatacccaaa agggg 35 <210> SEQ ID NO 88 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 88 ggccaagtgaagacccatgg aaggc 25 <210> SEQ ID NO 89 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 89 gcagtttccggattcgaagt gc 22 <210> SEQ ID NO 90 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 90 ccgctacgtctgtatta 17 <210> SEQ ID NO 91 <211> LENGTH: 17 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Oligonucleotide <400> SEQUENCE: 91 ataatggaag cacctga 17 <210>SEQ ID NO 92 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (22)..(22) <223> OTHER INFORMATION: N = T, G, C (6% each); A(82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(23)..(23) <223> OTHER INFORMATION: N = A, G, C (6% each); T (82%) <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (24)..(24) <223>OTHER INFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (25)..(25) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (26)..(26) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (27)..(28) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (29)..(29) <223> OTHERINFORMATION: N = T, G, C (6% each); A (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (30)..(31) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (32)..(32) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (33)..(33) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (34)..(34) <223> OTHERINFORMATION: N = A, T, G (6% each); C (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (35)..(35) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (36)..(36) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (37)..(38) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (39)..(39) <223> OTHERINFORMATION: N = A, T, G, C (25% each) <400> SEQUENCE: 92 agctatgctggtctcttctt annnnnnnnn nnnnnnnnna caattccatt ttttacttgg 60 <210> SEQ IDNO 93 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <400> SEQUENCE: 93 atccagttgg gtctctaaga aacaaaccgcgtaattaagc 40 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Oligonucleotide <400> SEQUENCE: 94 cctcaagggt tataacatcc 20<210> SEQ ID NO 95 <211> LENGTH: 55 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (19)..(19) <223> OTHER INFORMATION: N = A, T, C (6% each); G(82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(20)..(20) <223> OTHER INFORMATION: N = T, G, C (6% each); A (82%) <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>OTHER INFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (22)..(23) <223> OTHERINFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (24)..(24) <223> OTHERINFORMATION: N = A, T, G, C (25% each) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (25)..(25) <223> OTHER INFORMATION: N = A,G (8% each); T (2%); C (82%) <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (26)..(27) <223> OTHER INFORMATION: N = T, G, C (6%each); A (82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (28)..(28) <223> OTHER INFORMATION: N = A (82%); T (2%); G,C(8% each) <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(29)..(30) <223> OTHER INFORMATION: N = T, G, C (6% each); A (82%) <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (31)..(31) <223>OTHER INFORMATION: N = A, T, C (6% each); G (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (32)..(32) <223> OTHERINFORMATION: N = A, G, C (6% each); T (82%) <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (33)..(33) <223> OTHERINFORMATION: N = A, T, G, C (25% each) <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (34)..(34) <223> OTHER INFORMATION: N = A,G, C (6% each); T (82%) <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (35)..(35) <223> OTHER INFORMATION: N = T, G, C (6%each); A (82%) <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (36)..(36) <223> OTHER INFORMATION: N = A, G, C (1% each); T(97%) <400> SEQUENCE: 95 gtacaaaagc taagctttnn nnnnnnnnnn nnnnnncgaactatagctaa tacag 55 <210> SEQ ID NO 96 <211> LENGTH: 7 <212> TYPE: PRT<213> ORGANISM: Bacillus thuringiensis <400> SEQUENCE: 96 Ser Lys ArgSer Gln Asp Arg 1 5 <210> SEQ ID NO 97 <211> LENGTH: 1959 <212> TYPE:DNA <213> ORGANISM: Bacillus thuringiensis <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(1956) <400> SEQUENCE: 97 atg aat ccaaac aat cga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro AsnAsn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaattg caa act aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu LeuGln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tca acacta gaa gaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tctacg gaa gtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser ThrGlu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga atttct gtt gta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile SerVal Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggggca ctc act tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly AlaLeu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 aac act ata tgg cca agt gatgct gac cca tgg aag gct ttt atg gca 336 Asn Thr Ile Trp Pro Ser Asp AlaAsp Pro Trp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gataag aaa ata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp LysLys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cagggt ctt caa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tggaag aaa aca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa gat cgaata agg gaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg IleArg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atgccg tca ttt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met ProSer Phe Ala Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta cca aca tatgca caa gct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr AlaGln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gttttt gga gaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val PheGly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tatcat aga caa tta aaa ctt aca caa caa tac 720 Asp Val Ala Glu Phe Tyr HisArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gttaat tgg tat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val AsnTrp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gatgca tgg gtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp AlaTrp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gtatta gat cta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 att cgg tta tac tcaaaa ggg gtt aaa aca gaa cta aca aga gac att 912 Ile Arg Leu Tyr Ser LysGly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gat cca attttt tca ctt aat act ctt cag gag tat gga cca 960 Phe Thr Asp Pro Ile PheSer Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agtata gaa aac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag gggatt gaa ttt cat acg cgt ctt caa cct ggt tac ttt 1056 Tyr Leu Gln Gly IleGlu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 ggg aaa gat tctttc aat tat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser PheAsn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agt ata ggatct agt aag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly SerSer Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaacct gta caa aag cta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga actata gct aat aca gac gta gcg gct tgg ccg aat ggt aag 1248 Tyr Arg Thr IleAla Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 gta tat ttaggt gtt acg aaa gtt gat ttt agt caa tat gat gat caa 1296 Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaaact agt aca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta agtgca cag gat tct att gac caa tta ccg cca gaa aca aca 1392 His Val Ser AlaGln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa ccactt gaa aaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttctta atg cag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe LeuMet Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cataga agt gta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His ArgSer Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caactt cca gta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att attgaa ggt cca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile GluGly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tctagt aat tca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser SerAsn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gccttg tta caa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aactta cga ctt ttt gtg caa aat tca aac aat gat ttt ctt gtc atc 1776 Asn LeuArg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tacatt aat aaa act atg aat aaa gat gat gat tta aca tat caa aca 1824 Tyr IleAsn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 tttgat ctc gca act act aat tct aat atg ggg ttc tcg ggt gat aag 1872 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aatgaa ctt ata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn GluLeu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640tat ata gat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp LysIle Glu Phe Ile Pro Val Gln Leu 645 650 <210> SEQ ID NO 98 <211> LENGTH:652 <212> TYPE: PRT <213> ORGANISM: Bacillus thuringiensis <400>SEQUENCE: 98 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val ThrPro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu AlaAsp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe LeuArg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr ValLys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr GlnSer Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys AlaPhe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu GluTyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln AsnAsn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys ThrPro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp Arg Ile ArgGlu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met ProSer Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr TyrAla Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala GlnVal Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala GluPhe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr AspHis Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 GlySer Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His LeuPhe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln ProGly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr SerPro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr AspVal Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr LysVal Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser ThrGln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala GlnAsp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro LeuGlu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys PheLeu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 ThrHis Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala SerThr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp PheLeu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp LeuThr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met GlyPhe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser PheVal Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe IlePro Val Gln Leu 645 650 <210> SEQ ID NO 99 <211> LENGTH: 2000 <212>TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 99 ccatccatggcaaaccctaa caatcgttcc gaacacgaca ccatcaaggt tactccaaac 60 tctgagttgcaaactaatca caaccagtac ccattggctg acaatcctaa cagtactctt 120 gaggaacttaactacaagga gtttctccgg atgaccgaag atagctccac tgaggttctc 180 gataactctacagtgaagga cgctgttgga actggcatta gcgttgtggg acagattctt 240 ggagtggttggtgttccatt cgctggagct ttgaccagct tctaccagtc ctttctcaac 300 accatctggccttcagatgc tgatccctgg aaggctttca tggcccaagt ggaagtcttg 360 atcgataagaagatcgaaga gtatgccaag tctaaagcct tggctgagtt gcaaggtttg 420 cagaacaacttcgaggatta cgtcaacgca ctcaacagct ggaagaaaac tcccttgagt 480 ctcaggtctaagcgttccca ggaccgtatt cgtgaacttt tcagccaagc cgaatcccac 540 ttcagaaactccatgcctag ctttgccgtt tctaagttcg aggtgctctt cttgccaaca 600 tacgcacaagctgccaacac tcatctcttg cttctcaaag acgctcaggt gtttggtgag 660 gaatggggttactccagtga agatgttgcc gagttctacc gtaggcagct caagttgact 720 caacagtacacagaccactg cgtcaactgg tacaacgttg ggctcaatgg tcttagagga 780 tctacctacgacgcatgggt gaagttcaac aggtttcgta gagagatgac cttgactgtg 840 ctcgatcttatcgttctctt tccattctac gacattcgtc tttactccaa aggcgttaag 900 acagagctgaccagagacat cttcaccgat cccatcttcc tacttacgac cctgcagaaa 960 tacggtccaacttttctctc cattgagaac agcatcagga agcctcacct cttcgactat 1020 ctgcaaggcattgagtttca caccaggttg caacctggtt acttcggtaa ggattccttc 1080 aactactggagcggaaacta cgttgaaacc agaccatcca tcggatctag caagaccatc 1140 acttctccattctacggtga caagagcact gagccagtgc agaagttgag cttcgatggg 1200 cagaaggtgtatagaaccat cgccaatacc gatgttgcag cttggcctaa tggcaaggtc 1260 taccttggagttactaaagt ggacttctcc caatacgacg atcagaagaa cgagacatct 1320 actcaaacctacgatagtaa gaggaacaat ggccatgttt ccgcacaaga ctccattgac 1380 caacttccacctgaaaccac tgatgaacca ttggagaagg cttacagtca ccaacttaac 1440 tacgccgaatgctttctcat gcaagacagg cgtggcacca ttccgttctt tacatggact 1500 cacaggtctgtcgacttctt taacactatc gacgctgaga agattaccca acttcccgtg 1560 gtcaaggcttatgccttgtc cagcggagct tccatcattg aaggtccagg cttcaccggt 1620 ggcaacttgctcttccttaa ggagtccagc aactccatcg ccaagttcaa agtgacactt 1680 aactcagcagccttgctcca acgttacagg gttcgtatca gatacgcaag cactaccaat 1740 cttcgcctctttgtccagaa cagcaacaat gatttccttg tcatctacat caacaagact 1800 atgaacaaagacgatgacct cacctaccaa acattcgatc ttgccactac caatagtaac 1860 atgggattctctggtgacaa gaacgagctg atcataggtg ctgagagctt tgtctctaat 1920 gagaagatttacatagacaa gatcgagttc attccagttc aactctaata gatcccccgg 1980 gctgcaggaattcgatatca 2000 <210> SEQ ID NO 100 <211> LENGTH: 653 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 100 Met Ala AsnPro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr 1 5 10 15 Pro AsnSer Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp 20 25 30 Asn ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg 35 40 45 Met ThrGlu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys 50 55 60 Asp AlaVal Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val 65 70 75 80 ValGly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe 85 90 95 LeuAsn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met 100 105 110Ala Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys 115 120125 Ser Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp 130135 140 Tyr Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg145 150 155 160 Ser Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser GlnAla Glu 165 170 175 Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val SerLys Phe Glu 180 185 190 Val Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala AsnThr His Leu Leu 195 200 205 Leu Leu Lys Asp Ala Gln Val Phe Gly Glu GluTrp Gly Tyr Ser Ser 210 215 220 Glu Asp Val Ala Glu Phe Tyr Arg Arg GlnLeu Lys Leu Thr Gln Gln 225 230 235 240 Tyr Thr Asp His Cys Val Asn TrpTyr Asn Val Gly Leu Asn Gly Leu 245 250 255 Arg Gly Ser Thr Tyr Asp AlaTrp Val Lys Phe Asn Arg Phe Arg Arg 260 265 270 Glu Met Thr Leu Thr ValLeu Asp Leu Ile Val Leu Phe Pro Phe Tyr 275 280 285 Asp Ile Arg Leu TyrSer Lys Gly Val Lys Thr Glu Leu Thr Arg Asp 290 295 300 Ile Phe Thr AspPro Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly 305 310 315 320 Pro ThrPhe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe 325 330 335 AspTyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr 340 345 350Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr 355 360365 Arg Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly 370375 380 Asp Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys385 390 395 400 Val Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp ProAsn Gly 405 410 415 Lys Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser GlnTyr Asp Asp 420 425 430 Gln Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn 435 440 445 Gly His Val Ser Ala Gln Asp Ser Ile Asp GlnLeu Pro Pro Glu Thr 450 455 460 Thr Asp Glu Pro Leu Glu Lys Ala Tyr SerHis Gln Leu Asn Tyr Ala 465 470 475 480 Glu Cys Phe Leu Met Gln Asp ArgArg Gly Thr Ile Pro Phe Phe Thr 485 490 495 Trp Thr His Arg Ser Val AspPhe Phe Asn Thr Ile Asp Ala Glu Lys 500 505 510 Ile Thr Gln Leu Pro ValVal Lys Ala Tyr Ala Leu Ser Ser Gly Ala 515 520 525 Ser Ile Ile Glu GlyPro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu 530 535 540 Lys Glu Ser SerAsn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser 545 550 555 560 Ala AlaLeu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr 565 570 575 ThrAsn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val 580 585 590Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln 595 600605 Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp 610615 620 Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys625 630 635 640 Ile Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645650 <210> SEQ ID NO 101 <211> LENGTH: 2050 <212> TYPE: DNA <213>ORGANISM: Bacillus thuringiensis <400> SEQUENCE: 101 tggagctccaccgcggtggc ggccgctcta gaactagtgg atctaggcct ccatatgaac 60 cctaacaatcgttccgaaca cgacaccatc aaggttactc caaactctga gttgcaaact 120 aatcacaaccagtacccatt ggctgacaat cctaacagta ctcttgagga acttaactac 180 aaggagtttctccggatgac cgaagatagc tccactgagg ttctcgataa ctctacagtg 240 aaggacgctgttggaactgg cattagcgtt gtgggacaga ttcttggagt ggttggtgtt 300 ccattcgctggagctttgac cagcttctac cagtcctttc tcaacaccat ctggccttca 360 gatgctgatccctggaaggc tttcatggcc caagtggaag tcttgatcga taagaagatc 420 gaagagtatgccaagtctaa agccttggct gagttgcaag gtttgcagaa caacttcgag 480 gattacgtcaacgcactcaa cagctggaag aaaactccct tgagtctcag gtctaagcgt 540 tcccaggaccgtattcgtga acttttcagc caagccgaat cccacttcag aaactccatg 600 cctagctttgccgtttctaa gttcgaggtg ctcttcttgc caacatacgc acaagctgcc 660 aacactcatctcttgcttct caaagacgct caggtgtttg gtgaggaatg gggttactcc 720 agtgaagatgttgccgagtt ctaccatagg cagctcaagt tgactcaaca gtacacagac 780 cactgcgtcaactggtacaa cgttgggctc aatggtctta gaggatctac ctacgacgca 840 tgggtgaagttcaacaggtt tcgtagagag atgaccttga ctgtgctcga tcttatcgtt 900 ctctttccattctacgacat tcgtctttac tccaaaggcg ttaagacaga gctgaccaga 960 gacatcttcaccgatcccat cttctcactt aacaccctgc aggaatacgg tccaactttt 1020 ctctccattgagaacagcat caggaagcct cacctcttcg actatctgca aggcattgag 1080 tttcacaccaggttgcaacc tggttacttc ggtaaggatt ccttcaacta ctggagcgga 1140 aactacgttgaaaccagacc atccatcgga tctagcaaga ccatcacttc tccattctac 1200 ggtgacaagagcactgagcc agtgcagaag ttgagcttcg atgggcagaa ggtgtataga 1260 accatcgccaataccgatgt tgcagcttgg cctaatggca aggtctacct tggagttact 1320 aaagtggacttctcccaata cgacgatcag aagaacgaga catctactca aacctacgat 1380 agtaagaggaacaatggcca tgtttccgca caagactcca ttgaccaact tccacctgaa 1440 accactgatgaaccattgga gaaggcttac agtcaccaac ttaactacgc cgaatgcttt 1500 ctcatgcaagacaggcgtgg caccattccg ttctttacat ggactcacag gtctgtcgac 1560 ttctttaacactatcgacgc tgagaagatt acccaacttc ccgtggtcaa ggcttatgcc 1620 ttgtccagcggagcttccat cattgaaggt ccaggcttca ccggtggcaa cttgctcttc 1680 cttaaggagtccagcaactc catcgccaag ttcaaagtga cacttaactc agcagccttg 1740 ctccaacgttacagggttcg tatcagatac gcaagcacta ccaatcttcg cctctttgtc 1800 cagaacagcaacaatgattt ccttgtcatc tacatcaaca agactatgaa caaagacgat 1860 gacctcacctacaacacatt cgatcttgcc actaccaata gtaacatggg attctctggt 1920 gacaagaacgagctgatcat aggtgctgag agctttgtct ctaatgagaa gatttacata 1980 gacaagatcgagttcattcc agttcaactc taatagatcc cccgggctgc aggaattcga 2040 tatcaagctt2050 <210> SEQ ID NO 102 <211> LENGTH: 2280 <212> TYPE: DNA <213>ORGANISM: Bacillus thuringiensis <400> SEQUENCE: 102 ttaaaattaattttgtatac ttttcattgt aataatatga ttttaaaaac gaaaaagtgc 60 atatacaacttatcaggagg ggggggatgc acaaagaaga aaagaataag aagtgaatgt 120 ttataatgttcaatagtttt atgggaaggc attttatcag gtagaaagtt atgtattatg 180 ataagaatgggaggaagaaa aatgaatcca aacaatcgaa gtgaacatga tacgataaag 240 gttacacctaacagtgaatt gcaaactaac cataatcaat atcctttagc tgacaatcca 300 aattcaacactagaagaatt aaattataaa gaatttttaa gaatgactga agacagttct 360 acggaagtgctagacaactc tacagtaaaa gatgcagttg ggacaggaat ttctgttgta 420 gggcagattttaggtgttgt aggagttcca tttgctgggg cactcacttc attttatcaa 480 tcatttcttaacactatatg gccaagtgat gctgacccat ggaaggcttt tatggcacaa 540 gttgaagtactgatagataa gaaaatagag gagtatgcta aaagtaaagc tcttgcagag 600 ttacagggtcttcaaaataa tttcgaagat tatgttaatg cgttaaattc ctggaagaaa 660 acacctttaagtttgcgaag taaaagaagc caagatcgaa taagggaact tttttctcaa 720 gcagaaagtcattttcgtaa ttccatgccg tcatttgcag tttccaaatt cgaagtgctg 780 tttctaccaacatatgcaca agctgcaaat acacatttat tgctattaaa agatgctcaa 840 gtttttggagaagaatgggg atattcttca gaagatgttg ctgaatttta tcatagacaa 900 ttaaaacttacacaacaata cactgaccat tgtgttaatt ggtataatgt tggattaaat 960 ggtttaagaggttcaactta tgatgcatgg gtcaaattta accgttttcg cagagaaatg 1020 actttaactgtattagatct aattgtactt ttcccatttt atgatattcg gttatactca 1080 aaaggggttaaaacagaact aacaagagac atttttacgg atccaatttt ttcacttaat 1140 actcttcaggagtatggacc aacttttttg agtatagaaa actctattcg aaaacctcat 1200 ttatttgattatttacaggg gattgaattt catacgcgtc ttcaacctgg ttactttggg 1260 aaagattctttcaattattg gtctggtaat tatgtagaaa ctagacctag tataggatct 1320 agtaagacaattacttcccc attttatgga gataaatcta ctgaacctgt acaaaagcta 1380 agctttgatggacaaaaagt ttatcgaact atagctaata cagacgtagc ggcttggccg 1440 aatggtaaggtatatttagg tgttacgaaa gttgatttta gtcaatatga tgatcaaaaa 1500 aatgaaactagtacacaaac atatgattca aaaagaaaca atggccatgt aagtgcacag 1560 gattctattgaccaattacc gccagaaaca acagatgaac cacttgaaaa agcatatagt 1620 catcagcttaattacgcgga atgtttctta atgcaggacc gtcgtggaac aattccattt 1680 tttacttggacacatagaag tgtagacttt tttaatacaa ttgatgctga aaagattact 1740 caacttccagtagtgaaagc atatgccttg tcttcaggtg cttccattat tgaaggtcca 1800 ggattcacaggaggaaattt actattccta aaagaatcta gtaattcaat tgctaaattt 1860 aaagttacattaaattcagc agccttgtta caacgatatc gtgtaagaat acgctatgct 1920 tctaccactaacttacgact ttttgtgcaa aattcaaaca atgattttct tgtcatctac 1980 attaataaaactatgaataa agatgatgat ttaacatatc aaacatttga tctcgcaact 2040 actaattctaatatggggtt ctcgggtgat aagaatgaac ttataatagg agcagaatct 2100 ttcgtttctaatgaaaaaat ctatatagat aagatagaat ttatcccagt acaattgtaa 2160 ggagattttaaaatgttggg tgatggtcaa aatgaaagaa taggaaggtg aattttgatg 2220 gttaggaaagattcttttaa caaaagcaac atggaaaagt atacagtaca aatattaacc 2280 <210> SEQ IDNO 103 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <400> SEQUENCE: 103 taggcctcca tccatggcaa accctaacaa tc32 <210> SEQ ID NO 104 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticOligonucleotide <400> SEQUENCE: 104 tcccatcttc ctacttacga ccctgcagaaatacggtcca ac 42 <210> SEQ ID NO 105 <211> LENGTH: 28 <212> TYPE: DNA<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 105 gacctcacctaccaaacatt cgatcttg 28 <210> SEQ ID NO 106 <211> LENGTH: 25 <212> TYPE:DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 106 cgagttctaccgtaggcagc tcaag 25 <210> SEQ ID NO 107 <211> LENGTH: 1959 <212> TYPE:DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 107 atgaatccaaacaatcgaag tgaacatgat acgataaagg ttacacctaa cagtgaattg 60 caaactaaccataatcaata tcctttagct gacaatccaa attcaacact agaagaatta 120 aattataaagaatttttaag aatgactgaa gacagttcta cggaagtgct agacaactct 180 acagtaaaagatgcagttgg gacaggaatt tctgttgtag ggcagatttt aggtgttgta 240 ggagttccatttgctggggc actcacttca ttttatcaat catttcttaa cactatatgg 300 ccaagtgatgctgacccatg gaaggctttt atggcacaag ttgaagtact gatagataag 360 aaaatagaggagtatgctaa aagtaaagct cttgcagagt tacagggtct tcaaaataat 420 ttcgaagattatgttaatgc gttaaattcc tggaagaaaa cacctttaag tttgcgaagt 480 aaaagaagccaaggtcgaat aagggaactt ttttctcaag cagaaagtca ttttcgtaat 540 tccatgccgtcatttgcagt ttccaaattc gaagtgctgt ttctaccaac atatgcacaa 600 gctgcaaatacacatttatt gctattaaaa gatgctcaag tttttggaga agaatgggga 660 tattcttcagaagatgttgc tgaattctat cgtagacaat taaaacttac acaacaatac 720 actgaccattgtgttaattg gtataatgtt ggattaaatg gtttaagagg ttcaacttat 780 gatgcatgggtcaaatttaa ccgttttcgc agagaaatga ctttaactgt attagatcta 840 attgtacttttcccatttta tgatattcgg ttatactcaa aaggggttaa aacagaacta 900 acaagagacatttttacgga tccaattttt ttacttacta cgcttcagaa gtacggacca 960 acttttttgagtatagaaaa ctctattcga aaacctcatt tatttgatta tttacagggg 1020 attgaatttcatacgcgtct tcaacctggt tactttggga aagattcttt caattattgg 1080 tctggtaattatgtagaaac tagacctagt ataggatcta gtaagacaat tacttcccca 1140 ttttatggagataaatctac tgaacctgta caaaagctaa gctttgatgg acaaaaagtt 1200 tatcgaactatagctaatac agacgtagcg gcttggccga atggtaaggt atatttaggt 1260 gttacgaaagttgattttag tcaatatgat gatcaaaaaa atgaaactag tacacaaaca 1320 tatgattcaaaaagaaacaa tggccatgta agtgcacagg attctattga ccaattaccg 1380 ccagaaacaacagatgaacc acttgaaaaa gcatatagtc atcagcttaa ttacgcggaa 1440 tgtttcttaatgcaggaccg tcgtggaaca attccatttt ttacttggac acatagaagt 1500 gtagacttttttaatacaat tgatgctgaa aagattactc aacttccagt agtgaaagca 1560 tatgccttgtcttcaggtgc ttccattatt gaaggtccag gattcacagg aggaaattta 1620 ctattcctaaaagaatctag taattcaatt gctaaattta aagttacatt aaattcagca 1680 gccttgttacaacgatatcg tgtaagaata cgctatgctt ctaccactaa cttacgactt 1740 tttgtgcaaaattcaaacaa tgattttctt gtcatctaca ttaataaaac tatgaataaa 1800 gatgatgatttaacatatca aacatttgat ctcgcaacta ctaattctaa tatggggttc 1860 tcgggtgataagaatgaact tataatagga gcagaatctt tcgtttctaa tgaaaaaatc 1920 tatatagataagatagaatt tatcccagta caattgtaa 1959 <210> SEQ ID NO 108 <211> LENGTH:652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 108Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 1015 Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 2530 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 4045 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 5560 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 7075 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 8590 95 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe GluAsp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 Lys Arg Ser Gln Gly Arg Ile Arg Glu Leu PheSer Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe AlaVal Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe GlyGlu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr ArgArg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys ValAsn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr TyrAsp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 PheThr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly Pro 305 310 315320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu ThrArg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe TyrGly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp GlyGln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp PheSer Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser IleAsp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys AlaTyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg SerVal Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln LeuPro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile IleGlu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 GluSer Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile580 585 590 Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr GlnThr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser GlyAsp Lys 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser AsnGlu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 <210> SEQ ID NO 109 <211> LENGTH: 649 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <400> SEQUENCE: 109 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Ala Thr Glu 1 5 10 15 Asn Asn Glu Val SerAsn Asn His Ala Gln Tyr Pro Leu Ala Asp Thr 20 25 30 Pro Thr Leu Glu GluLeu Asn Tyr Lys Glu Phe Leu Arg Arg Thr Thr 35 40 45 Asp Asn Asn Val GluAla Leu Asp Ser Ser Thr Thr Lys Asp Ala Ile 50 55 60 Gln Lys Gly Ile SerIle Ile Gly Asp Leu Leu Gly Val Val Gly Phe 65 70 75 80 Pro Tyr Gly GlyAla Leu Val Ser Phe Tyr Thr Asn Leu Leu Asn Thr 85 90 95 Ile Trp Pro GlyGlu Asp Pro Leu Lys Ala Phe Met Gln Gln Val Glu 100 105 110 Ala Leu IleAsp Gln Lys Ile Ala Asp Tyr Ala Lys Asp Lys Ala Thr 115 120 125 Ala GluLeu Gln Gly Leu Lys Asn Val Phe Lys Asp Tyr Val Ser Ala 130 135 140 LeuAsp Ser Trp Asp Lys Thr Pro Leu Thr Leu Arg Asp Gly Arg Ser 145 150 155160 Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg 165170 175 Arg Ser Met Pro Ser Phe Ala Val Ser Gly Tyr Glu Val Leu Phe Leu180 185 190 Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu Leu LysAsp 195 200 205 Ala Gln Ile Tyr Gly Thr Asp Trp Gly Tyr Ser Thr Asp AspLeu Asn 210 215 220 Glu Phe His Thr Lys Gln Lys Asp Leu Thr Ile Glu TyrThr Asn His 225 230 235 240 Cys Ala Lys Trp Tyr Lys Ala Gly Leu Asp LysLeu Arg Gly Ser Thr 245 250 255 Tyr Glu Glu Trp Val Lys Phe Asn Arg TyrArg Arg Glu Met Thr Leu 260 265 270 Thr Val Leu Asp Leu Ile Thr Leu PhePro Leu Tyr Asp Val Arg Thr 275 280 285 Tyr Thr Lys Gly Val Lys Thr GluLeu Thr Arg Asp Val Leu Thr Asp 290 295 300 Pro Ile Val Ala Val Asn AsnMet Asn Gly Tyr Gly Thr Thr Phe Ser 305 310 315 320 Asn Ile Glu Asn TyrIle Arg Lys Pro His Leu Phe Asp Tyr Leu His 325 330 335 Ala Ile Gln PheHis Ser Arg Leu Gln Pro Gly Tyr Phe Gly Thr Asp 340 345 350 Ser Phe AsnTyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Ser Ser Ile 355 360 365 Gly SerAsp Glu Ile Ile Arg Ser Pro Phe Tyr Gly Asn Lys Ser Thr 370 375 380 LeuAsp Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Phe Arg Ala 385 390 395400 Val Ala Asn Gly Asn Leu Ala Val Trp Pro Val Gly Thr Gly Gly Thr 405410 415 Lys Ile His Ser Gly Val Thr Lys Val Gln Phe Ser Gln Tyr Asn Asp420 425 430 Arg Lys Asp Glu Val Arg Thr Gln Thr Tyr Asp Ser Lys Arg AsnVal 435 440 445 Gly Gly Ile Val Phe Asp Ser Ile Asp Gln Leu Pro Pro IleThr Thr 450 455 460 Asp Glu Ser Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Val Arg 465 470 475 480 Cys Phe Leu Leu Gln Gly Gly Arg Gly Ile IlePro Val Phe Thr Trp 485 490 495 Thr His Lys Ser Val Asp Phe Tyr Asn ThrLeu Asp Ser Glu Lys Ile 500 505 510 Thr Gln Ile Pro Phe Val Lys Ala PheIle Leu Val Asn Ser Thr Ser 515 520 525 Val Val Ala Gly Pro Gly Phe ThrGly Gly Asp Ile Ile Lys Cys Thr 530 535 540 Asn Gly Ser Gly Leu Thr LeuTyr Val Thr Pro Ala Pro Asp Leu Thr 545 550 555 560 Tyr Ser Lys Thr TyrLys Ile Arg Ile Arg Tyr Ala Ser Thr Ser Gln 565 570 575 Val Arg Phe GlyIle Asp Leu Gly Ser Tyr Thr His Ser Ile Ser Tyr 580 585 590 Phe Asp LysThr Met Asp Lys Gly Asn Thr Leu Thr Tyr Asn Ser Phe 595 600 605 Asn LeuSer Ser Val Ser Arg Pro Ile Glu Ile Ser Gly Gly Asn Lys 610 615 620 IleGly Val Ser Val Gly Gly Ile Gly Ser Gly Asp Glu Val Tyr Ile 625 630 635640 Asp Lys Ile Glu Phe Ile Pro Met Asp 645 <210> SEQ ID NO 110 <211>LENGTH: 652 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Recombinant delta endotoxin <400>SEQUENCE: 110 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys ValThr Pro 1 5 10 15 Asn Ser Glu Leu Pro Thr Asn His Asn Gln Tyr Pro LeuAla Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu PheLeu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile LeuGly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe TyrGln Ser Phe Leu 85 90 95 Asp Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp LysAla Phe Met Ala 100 105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile GluGlu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu GlnAsn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys LysThr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg Ser Gln Asp Arg IleArg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser MetPro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro ThrTyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp AlaGln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val AlaGlu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 ThrAsp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275280 285 Val Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr GlyPro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro HisLeu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu GlnPro Gly Tyr Ser 340 345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly AsnTyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile ThrSer Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys LeuSer Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn ThrAsp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 Ile Tyr Phe Gly Val ThrLys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr SerThr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 His Val Gly AlaGln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu ProLeu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 CysPhe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn SerAla 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr AlaSer Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn AspPhe Ile Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met Asn Ile Asp Asp AspLeu Thr Tyr Gln Thr 595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn MetGly Phe Ser Gly Asp Thr 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu PheIle Pro Val Gln Leu 645 650 <210> SEQ ID NO 111 <211> LENGTH: 652 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Recombinant delta endotoxin <400> SEQUENCE: 111 Met Asn ProAsn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 GlyVal Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 AsnThr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser LysPhe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu TrpGly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln LeuLys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala TrpVal Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr SerLys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp ProIle Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro AsnGly Lys 405 410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser LysArg Asn Asn Gly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser HisGln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp PhePhe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val ValLys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser AsnSer Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 AsnLeu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650<210> SEQ ID NO 112 <211> LENGTH: 659 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Recombinantdelta endotoxin <400> SEQUENCE: 112 Met Ile Arg Met Gly Gly Arg Lys MetAsn Pro Asn Asn Arg Ser Glu 1 5 10 15 Tyr Asp Thr Ile Lys Val Thr ProAsn Ser Glu Leu Pro Thr Asn His 20 25 30 Asn Gln Tyr Pro Leu Ala Asp AsnPro Asn Ser Thr Leu Glu Glu Leu 35 40 45 Asn Tyr Lys Glu Phe Leu Arg MetThr Ala Asp Asn Ser Thr Glu Val 50 55 60 Leu Asp Ser Ser Thr Val Lys AspAla Val Gly Thr Gly Ile Ser Val 65 70 75 80 Val Gly Gln Ile Leu Gly ValVal Gly Val Pro Phe Ala Gly Ala Leu 85 90 95 Thr Ser Phe Tyr Gln Ser PheLeu Asn Ala Ile Trp Pro Ser Asp Ala 100 105 110 Asp Pro Trp Lys Ala PheMet Ala Gln Val Glu Val Leu Ile Asp Lys 115 120 125 Lys Ile Glu Glu TyrAla Lys Ser Lys Ala Leu Ala Glu Leu Gln Gly 130 135 140 Leu Gln Asn AsnPhe Glu Asp Tyr Val Asn Ala Leu Asp Ser Trp Lys 145 150 155 160 Lys AlaPro Val Asn Leu Arg Ser Arg Arg Ser Gln Asp Arg Ile Arg 165 170 175 GluLeu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser 180 185 190Phe Ala Val Ser Lys Phe Glu Val Leu Phe Leu Pro Thr Tyr Ala Gln 195 200205 Ala Ala Asn Thr His Leu Leu Leu Leu Lys Asp Ala Gln Val Phe Gly 210215 220 Glu Glu Trp Gly Tyr Ser Ser Glu Asp Ile Ala Glu Phe Tyr Gln Arg225 230 235 240 Gln Leu Lys Leu Thr Gln Gln Tyr Thr Asp His Cys Val AsnTrp Tyr 245 250 255 Asn Val Gly Leu Asn Ser Leu Arg Gly Ser Thr Tyr AspAla Trp Val 260 265 270 Lys Phe Asn Arg Phe Arg Arg Glu Met Thr Leu ThrVal Leu Asp Leu 275 280 285 Ile Val Leu Phe Pro Phe Tyr Asp Val Arg LeuTyr Ser Lys Gly Val 290 295 300 Lys Thr Glu Leu Thr Arg Asp Ile Phe ThrAsp Pro Ile Phe Thr Leu 305 310 315 320 Asn Ala Leu Gln Glu Tyr Gly ProThr Phe Ser Ser Ile Glu Asn Ser 325 330 335 Ile Arg Lys Pro His Leu PheAsp Tyr Leu Arg Gly Ile Glu Phe His 340 345 350 Thr Arg Leu Arg Pro GlyTyr Ser Gly Lys Asp Ser Phe Asn Tyr Trp 355 360 365 Ser Gly Asn Tyr ValGlu Thr Arg Pro Ser Ile Gly Ser Asn Asp Thr 370 375 380 Ile Thr Ser ProPhe Tyr Gly Asp Lys Ser Ile Glu Pro Ile Gln Lys 385 390 395 400 Leu SerPhe Asp Gly Gln Lys Val Tyr Arg Thr Ile Ala Asn Thr Asp 405 410 415 IleAla Ala Phe Pro Asp Gly Lys Ile Tyr Phe Gly Val Thr Lys Val 420 425 430Asp Phe Ser Gln Tyr Asp Asp Gln Lys Asn Glu Thr Ser Thr Gln Thr 435 440445 Tyr Asp Ser Lys Arg Tyr Asn Gly Tyr Leu Gly Ala Gln Asp Ser Ile 450455 460 Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys Ala Tyr465 470 475 480 Ser His Gln Leu Asn Tyr Ala Glu Cys Phe Leu Met Gln AspArg Arg 485 490 495 Gly Thr Ile Pro Phe Phe Thr Trp Thr His Arg Ser ValAsp Phe Phe 500 505 510 Asn Thr Ile Asp Ala Glu Lys Ile Thr Gln Leu ProVal Val Lys Ala 515 520 525 Tyr Ala Leu Ser Ser Gly Ala Ser Ile Ile GluGly Pro Gly Phe Thr 530 535 540 Gly Gly Asn Leu Leu Phe Leu Lys Glu SerSer Asn Ser Ile Ala Lys 545 550 555 560 Phe Lys Val Thr Leu Asn Ser AlaAla Leu Leu Gln Arg Tyr Arg Val 565 570 575 Arg Ile Arg Tyr Ala Ser ThrThr Asn Leu Arg Leu Phe Val Gln Asn 580 585 590 Ser Asn Asn Asp Phe LeuVal Ile Tyr Ile Asn Lys Thr Met Asn Ile 595 600 605 Asp Gly Asp Leu ThrTyr Gln Thr Phe Asp Phe Ala Thr Ser Asn Ser 610 615 620 Asn Met Gly PheSer Gly Asp Thr Asn Asp Phe Ile Ile Gly Ala Glu 625 630 635 640 Ser PheVal Ser Asn Glu Lys Ile Tyr Ile Asp Lys Ile Glu Phe Ile 645 650 655 ProVal Gln <210> SEQ ID NO 113 <211> LENGTH: 652 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Recombinant delta endotoxin <400> SEQUENCE: 113 Met Ile Arg Lys Gly GlyArg Lys Met Asn Pro Asn Asn Arg Ser Glu 1 5 10 15 His Asp Thr Ile LysThr Thr Glu Asn Asn Glu Val Pro Thr Asn His 20 25 30 Val Gln Tyr Pro LeuAla Glu Thr Pro Asn Pro Thr Leu Glu Asp Leu 35 40 45 Asn Tyr Lys Glu PheLeu Arg Met Thr Ala Asp Asn Asn Thr Glu Ala 50 55 60 Leu Asp Ser Ser ThrThr Lys Asp Val Ile Gln Lys Gly Ile Ser Val 65 70 75 80 Val Gly Asp LeuLeu Gly Val Val Gly Phe Pro Phe Gly Gly Ala Leu 85 90 95 Val Ser Phe TyrThr Asn Phe Leu Asn Thr Ile Trp Pro Ser Glu Asp 100 105 110 Pro Trp LysAla Phe Met Glu Gln Val Glu Ala Leu Met Asp Gln Lys 115 120 125 Ile AlaAsp Tyr Ala Lys Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu 130 135 140 GlnAsn Asn Val Glu Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys 145 150 155160 Asn Pro Val Ser Ser Arg Asn Pro His Ser Gln Gly Arg Ile Arg Glu 165170 175 Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser Phe180 185 190 Ala Ile Ser Gly Tyr Glu Val Leu Phe Leu Thr Thr Tyr Ala GlnAla 195 200 205 Ala Asn Thr His Leu Phe Leu Leu Lys Asp Ala Gln Ile TyrGly Glu 210 215 220 Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala Glu Phe TyrLys Arg Gln 225 230 235 240 Leu Lys Leu Thr Gln Glu Tyr Thr Asp His CysVal Lys Trp Tyr Asn 245 250 255 Val Gly Leu Asp Lys Leu Arg Gly Ser SerTyr Glu Ser Trp Val Asn 260 265 270 Phe Asn Arg Tyr Arg Arg Glu Met ThrLeu Thr Val Leu Asp Leu Ile 275 280 285 Ala Leu Phe Pro Leu Tyr Asp ValArg Leu Tyr Pro Lys Glu Val Lys 290 295 300 Thr Glu Leu Thr Arg Asp ValLeu Thr Asp Pro Ile Val Gly Val Asn 305 310 315 320 Asn Leu Arg Gly TyrGly Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile 325 330 335 Arg Lys Pro HisLeu Phe Asp Tyr Leu His Arg Ile Gln Phe His Thr 340 345 350 Arg Phe GlnPro Gly Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser 355 360 365 Gly AsnTyr Val Ser Thr Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile 370 375 380 ThrSer Pro Phe Tyr Gly Asn Lys Ser Ser Glu Pro Val Gln Asn Leu 385 390 395400 Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala Val Ala Asn Thr Asn Leu 405410 415 Ala Val Trp Pro Ser Ala Val Tyr Ser Gly Val Thr Lys Val Glu Phe420 425 430 Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala Ser Thr Gln Thr TyrAsp 435 440 445 Ser Lys Arg Asn Val Gly Ala Val Ser Trp Asp Ser Ile AspGln Leu 450 455 460 Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys Gly TyrSer His Gln 465 470 475 480 Leu Asn Tyr Val Met Cys Phe Leu Met Gln GlySer Arg Gly Thr Ile 485 490 495 Pro Val Leu Thr Trp Thr His Lys Ser ValAsp Phe Phe Asn Met Ile 500 505 510 Asp Ser Lys Lys Ile Thr Gln Leu ProLeu Val Lys Ala Tyr Lys Leu 515 520 525 Gln Ser Gly Ala Ser Val Val AlaGly Pro Arg Phe Thr Gly Gly Asp 530 535 540 Ile Ile Gln Cys Thr Glu AsnGly Ser Ala Ala Thr Ile Tyr Val Thr 545 550 555 560 Pro Asp Val Ser TyrSer Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala 565 570 575 Ser Thr Ser GlnIle Thr Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe 580 585 590 Asn Gln TyrTyr Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr 595 600 605 Tyr AsnSer Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser 610 615 620 GlyAsn Asn Leu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys 625 630 635640 Val Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Asn 645 650

What is claimed is:
 1. An isolated nucleic acid segment comprising aδ-endotoxin gene encoding a Cry* polypeptide selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:100, and SEQ ID NO:108.
 2. The polynucleotide of claim1, further defined as comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:99, SEQ ID NO:99, and SEQ ID NO:107.
 3. Thepolynucleotide of claim 1, further characterized as DNA, cDNA, rRNA, ormRNA.
 4. A nucleic acid segment of from about 1800 to about 18,000nucleotides in length comprising a gene encoding a modified Cry3*δ-endotoxin polypeptide having improved insect activity or enhancedinsect specificity against a target insect when compared to theunmodified Cry3 polypeptide.
 5. The nucleic acid segment of claim 4,wherein said nucleic acid segment is from about 2000 to about 10,000nucleotides in length.
 6. The nucleic acid segment of claim 5, whereinsaid nucleic acid segment is from about 3000 to about 8,000 nucleotidesin length.
 7. The nucleic acid segment of claim 4, wherein said gene isoperably linked to a promoter, said promoter expressing said gene. 8.The nucleic acid segment of claim 4, comprised within a vector.
 9. Thenucleic acid segment of claim 4, comprised within a transgenic plant.10. The nucleic acid segment of claim 4, wherein said modified Cry3*crystal protein is obtained by random or site-specific mutagenesis ofthe nucleic acid segment which encodes it.
 11. A nucleic acid segment offrom about 1800 to about 18,000 nucleotides in length comprising a cry3*gene encoding a modified Cry3* crystal protein, wherein said modifiedCry3* crystal protein has improved insecticidal activity or specificitywhen compared to a native unmodified Cry3 crystal protein.
 12. Thenucleic acid segment of claim 11, wherein said modified Cry3* crystalprotein is obtained by random or site-specific mutagenesis of thenucleic acid segment which encodes it.
 13. The nucleic acid segment ofclaim 12, wherein said mutagenesis results in one or more nucleotidesubstitutions, deletions, or insertions.
 14. The nucleic acid segment ofclaim 13, wherein said mutagenesis results in one or more amino acidsubstitutions, deletions, or insertions in the encoded Cry3* protein.15. The nucleic acid segment claim 11, wherein said gene encodes aCry3Bb* crystal protein selected from the group consisting of Cry3Bb-60,Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225,Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230,Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235,Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241,Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036, Cry3Bb.11046,Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057, Cry3Bb.11058, Cry3Bb.11081,Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11095, andCry3Bb.11098.
 16. The nucleic acid segment of claim 11, wherein saidcry3* gene is further defined as a cry3A*, cry3B*, or cry3C* gene. 17.The nucleic acid segment of claim 16, wherein said cry3B* gene isfurther defined as a cry3Bb* gene.
 18. The nucleic acid segment of claim17, wherein said cry3Bb* gene is further defined as a cry3Bb-60,cry3Bb.11221, cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225,cry3Bb.11226, cry3Bb.11227, cry3Bb.11228, cry3Bb.11229, cry3Bb.11230,cry3Bb.11231, cry3Bb.11232, cry3Bb.11233, cry3Bb.11234, cry3Bb.11235,cry3Bb.11236, cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241,cry3Bb.11242, cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046,cry3Bb.11048, cry3Bb.11051, cry3Bb.11057, cry3Bb.11058, cry3Bb.11081,cry3Bb.11082, cry3Bb.11083, cry3Bb.11084, cry3Bb.11095 or cry3Bb.11098gene.
 19. A method of preparing a modified Cry3* polypeptide havingimproved insecticidal activity or specificity, comprising the steps of:(a) providing a nucleic acid segment comprising a gene which encodes aCry3* polypeptide, wherein said gene is operably linked to a promoterwhich expresses said gene; (b) introducing said nucleic acid segmentinto a vector; (c) transforming a host cell with said vector; and (d)culturing said host cell under conditions effective to allow expressionof the modified Cry3* polypeptide.
 20. The method of claim 19, whereinsaid gene encodes a modified Cry3A*, Cry3B*, or Cry3C* polypeptide. 21.The method of claim 19, wherein said host cell is a plant, animal,viral, fungal, archaebacterial, eubacterial, or cyanobacterial hostcell.
 22. The method of claim 19, wherein said gene comprises a cry3A*,cry3B*, or cry3C* gene.
 23. The method of claim 22, wherein said cry3B*gene is selected from the group consisting of a cry3Bb-60, cry3Bb.11221,cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225, cry3Bb.11226,cry3Bb.11227, cry3Bb.11228, cry3Bb.11229, cry3Bb.11230, cry3Bb.11231,cry3Bb.11232, cry3Bb.11233, cry3Bb.11234, cry3Bb.11235, cry3Bb.11236,cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241, cry3Bb.11242,cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046, cry3Bb.11048,cry3Bb.11051, cry3Bb.11057, cry3Bb.11058, cry3Bb.11081, cry3Bb.11082,cry3Bb.11083, cry3Bb.11084, cry3Bb.11095, and cry3Bb.11098.
 24. A vectorcomprising a nucleic acid segment encoding a B. thuringiensis Cry3*polypeptide, wherein said polypeptide has at least one modified aminoacid sequence in one or more domains of said polypeptide, relative to awild-type unmodified Cry3 polypeptide; and further wherein said modifiedamino acid sequence confers to said polypeptide an insecticidal activitygreater than, or an insecticidal specificity broader than, that of saidwild-type unmodified Cry3 polypeptide.
 25. The vector of claim 24,wherein said Cry3* polypeptide is selected from the group consisting ofCry3Bb-60, Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224,Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228, Cry3Bb.11229,Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234,Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239,Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036,Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057, Cry3Bb.11058,Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11095,and Cry3Bb.11098.
 26. The vector of claim 25, wherein said Cry3*polypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:100, and SEQ ID NO:108.
 27. The vector of claim 26,wherein said Cry3* polypeptide is encoded by a nucleic acid segmentselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, and SEQ ID NO:107. 28.The vector of claim 24, transformed and replicated in a prokaryotic oreukaryotic host.
 29. The vector of claim 28, comprised within a plantcell.
 30. The vector of claim 24, wherein said polypeptide is encoded bya nucleic acid segment contained within a plasmid selected from thegroup consisting of pEG1707, pEG1708, pEG1709, pEG1710, pEG1711,pEG1712, pEG1713, pEG1714, pEG1715, pEG1716, pEG1717, pEG1718, pEG1719,pEG1720, pEG1721, pEG1722, pEG1723, pEG1724, pEG1725, pEG1726, pEG1727,pEG1041, pEG1046, pEG1047, pEG1052, pEG1054, pEG1057, pEG1062, pEG1063,pEG1084, pEG1085, pEG1086, pEG1087, pEG1095, and pEG1098.
 31. The vectorof claim 24, further defined as a cosmid, plasmid, phagemid,baculovirus, artificial chromosome, or viral vector.
 32. A vectorcomprising the nucleic acid segment of claim
 1. 33. A transformed hostcell which expresses the polynucleotide of claim
 1. 34. A method ofpreparing a Coleopteran-resistant transgenic plant, comprising the stepsof: (a) obtaining a nucleic acid segment comprising a gene which encodesa modified Cry3* polypeptide, wherein said gene is operably linked to apromoter which expresses said gene; (b) introducing said nucleic acidsegment into a vector; (c) transforming a plant cell with said vector;and (d) generating from said plant cell a transgenic plant whichexpresses said modified Cry3* polypeptide.
 35. The method of claim 34,wherein said gene comprises a δ-endotoxin gene encoding a Cry3*polypeptide selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, and SEQID NO:108.